Pathophysiology of primary hyperparathyroidism

Parathyroid gland is the overall regulatory organ within the systemic calcium homeostasis. Through cell surface bound calcium-sensing receptors external calcium inversely regulates release of parathyroid hormone (PTH). This mechanism, which is voltage independent and most sensitive around physiologic calcium concentrations, is regulated through a 120 kDa calcium sensing receptor, CaR. Inherited inactivation of this receptor is the cause for familial hypocalciuric hypercalcemia (FHH). Parallel research identified the 550 kDa glycoprotein megalin, which also is expressed on the parathyroid cell surface, as another potential calcium sensing protein. Although this protein expresses numerous calcium binding sites on its external domain, its main function may be calcium sensitive binding and uptake of steroid hormones, such as 25-OH-vitamin D3 (bound to vitamin D binding protein) and retinol. In hyperparathyroidism (HPT), excessive PTH is secreted and the calcium sensitivity of the cells reduced, i.e. the set-point, defined as the external calcium concentration at which half-maximal inhibition of PTH release occurs, shifted to the right. Pathological cells have reduced expression of both CaR and megalin, and reduced amount of intracellular lipids, possibly including stored steroid hormones. A number of possible genetic disturbances have been identified, indicating multifactorial reasons for the disease. In postmenopausal women, however, the individual group with highest incidence of disease, a causal relation to reduced effect of vitamin D is possible. An incipient renal insufficiency with age, lack of sunshine in the Northern Hemisphere, and an association to the baT haplotype of the vitamin D receptor supports this theory. This review summarizes data on regulation of PTH release, dysregulation in HPT, as well as proliferation of parathyroid cells.     

Calcium Sensing Receptor Absence Delays Postnatal Brain Development via Direct and Indirect Mechanisms

Calcium sensing receptor (CaSR) is implicated in the establishment of neural connections and myelin formation. However, its contribution to brain development remains unclear. We addressed this issue by analyzing brain phenotype in postnatal CaSR null mice, a model of human neonatal severe hyperparathyroidism. One- and 2-week-old CaSR null mice exhibited decreased brain weight and size with a developmental delay in expression of proliferating cell nuclear antigen. Neuronal and glial differentiation markers, neuronal specific nuclear protein, glial fibrillary acidic protein, and myelin basic protein, were also decreased compared with age-matched wild-type littermates. Moreover, deletion of the parathyroid hormone gene that corrects hyperparathyroidism, hypercalcemia, hypophosphatemia, and whole-body growth retardation normalized brain cell proliferation, but not differentiation, in CaSR null mice. Cultured neural stem cells (NSCs) derived from the subventricular zones of CaSR null neonatal mice exhibited normal proliferation capacity but decreased differentiation capacity, compared with wild-type controls. These results demonstrate that direct effects of CaSR absence impair NSC differentiation, while secondary effects of parathyroid hormone-related endocrine abnormalities impair NSC proliferation, both of which contribute to delayed brain development in CaSR null newborn mice.     

Regulator of G protein signaling 5 is highly expressed in parathyroid tumors and inhibits signaling by the calcium-sensing receptor

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.     

Alterations in the sensing and transport of phosphate and calcium by differentiating chondrocytes

During endochondral bone formation and fracture healing, cells committed to chondrogenesis undergo a temporally restricted program of differentiation that is characterized by sequential changes in their phenotype and gene expression. This results in the manufacture, remodeling, and mineralization of a cartilage template on which bone is laid down. Articular chondrocytes undergo a similar but restricted differentiation program that does not proceed to mineralization, except in pathologic conditions such as osteoarthritis. The pathogenesis of disorders of cartilage development and metabolism, including osteochondrodysplasia, fracture non-union, and osteoarthritis remain poorly defined. We used the CFK2 model to examine the potential roles of phosphate and calcium ions in the regulatory pathways that mediate chondrogenesis and cartilage maturation. Differentiation was monitored over a 4-week period using a combination of morphological, biochemical, and molecular markers that have been characterized in vivo and in vitro. CFK2 cells expressed the type III sodium-dependent phosphate transporters Glvr-1 and Ram-1, as well as a calcium-sensing mechanism. Regulated expression and activity of Glvr-1 by extracellular phosphate and parathyroid hormone-related protein was restricted to an early stage of CFK2 differentiation, as evidenced by expression of type II collagen, proteoglycan, and Ihh. On the other hand, regulated expression and activity of a calcium-sensing receptor by extracellular calcium was most evident after 2 weeks of differentiation, concomitant with an increase in type X collagen expression, alkaline phosphatase activity and parathyroid hormone/parathyroid hormone-related protein receptor expression. On the basis of these temporally restricted changes in the sensing and transport of phosphate and calcium, we predict that extracellular phosphate plays a role in the commitment of chondrogenic cells to differentiation, whereas extracellular calcium plays a role at a later stage in their differentiation program.     

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.     

Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.     

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

Objective

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr^db/lb) to determine the role of leptin in skeletal muscle.

Methods and Findings

Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion

These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.     

Abnormalities of the hypothalamo-pituitary-thyroid axis in the pro-opiomelanocortin deficient mouse

The melanocortin system is an important regulator of body weight and the hypothalamo-pituitary-thyroid (HPT) axis. The pro-opiomelanocortin (POMC)-null mouse, deficient in all POMC-derived peptides, including alpha-melanocyte stimulating hormone (alpha-MSH), has an obese phenotype. We studied the HPT axis of POMC-null mice, which has not been previously investigated. Because alpha-MSH has a stimulatory effect on the HPT axis, we hypothesised that these mice would have a down-regulated thyroid axis, consistent with a recent study of POMC-null humans. The activity of the HPT axis was studied by collecting blood, pituitaries and hypothalami from ad libitum fed, adult POMC-null, heterozygous and wild-type mice. POMC-null mice had significantly elevated plasma total T(4) (TT(4)) and free T(3) (fT(3)) with reduced plasma thyroid stimulating hormone (TSH), pituitary TSH content and hypothalamic thyrotrophin stimulating hormone (TRH) content compared to wild-type mice. No significant differences between heterozygous and wild-type mice were observed. POMC-null mice have an abnormal HPT axis, which may contribute to their hyperphagia and obesity. These abnormalities are in contrast to those observed in POMC-null humans. These findings support a role for the melanocortin system in the regulation of the HPT axis.     

Recombinant Human Parathyroid Hormone Therapy (1-34) In an Adult Patient with a Gain-of-Function Mutation in the Calcium-Sensing Receptor-a Case Report

ObjectiveTo describe a case of hypocalcemia in a patient with a gain-of-function mutation in the calcium-sensing receptor that was undetected until adulthood and successfully treated with recombinant parathyroid hormone.MethodsThe clinical findings, laboratory data, and a review of the pertinent literature are presented.ResultsA 55-year-old woman was hospitalized and seen by the endocrinology consult service for hypocalcemia that was refractory to repeated doses of intravenous calcium gluconate. She expressed concern about chronic leg muscle cramps and paresthesias of the lips and fingertips. In addition, she had no history of neck surgery, neck irradiation, or any autoimmune disease. She was a well-appearing female with no dysmorphic features or skin changes. Laboratory tests revealed hypocalcemia, hyperphosphatemia, hypomagnesemia, and hypovitamino-sis D. Her parathyroid hormone concentration (PTH) was low at 14.2 pg/mL. Her PTH and calcium concentrations remained low despite repletion of magnesium and treatment with calcitriol and oral calcium replacement. A 24-hour collection for urinary calcium showed inappropriate hypercalciuria. Medical records showed her hypocalcemia to be chronic. Additionally, several family members had also complained of muscle cramps. A congenital cause of her hypoparathyroidism was considered, and genetic testing confirmed heterozygosity for a gain-of-function mutation in the calcium-sensing receptor gene associated with autosomal dominant familial isolated hypoparathyroidism (ADH). Treatment with subcutaneous recombinant human parathyroid hormone teriparatide (rhPTH [1-34]) 20 mcg twice daily for three days normalized her calcium and phosphorus concentrations.ConclusionrhPTH (1-34) is an effective treatment for patients with hypoparathyroidism due to gain-of-function mutations in the calcium-sensing receptor. ADH can be insidious in presentation and the diagnosis can be missed unless there is a high index of suspicion. (Endocr Pract. 2013;19:e24-e28)     

Inhibition of PTH Secretion by Interleukin-1β in Bovine Parathyroid Glandsin VitroIs Associated with an Up-Regulation of the Calcium-Sensing Receptor mRNA

The principal regulator of parathyroid hormone (PTH) secretion is ionized calcium, but other factors are also known to modulate PTH secretion, such as vitamin D, estrogen, and recently inorganic phosphate. Interleukin-1 (IL-1) possesses a wide variety of biological activities and is produced by leukocytes as well as by various other cells including cells from endocrine tissues and might play a role as a paracrine factor in the control of PTH secretion. We investigated the effectin vitroof IL-1β on PTH release, PTHmRNA and the mRNA for the extracellular calcium-sensing receptor (CaR) levels in preparations of bovine parathyroid cells. PTH secretion from cultured parathyroid tissue slices was significantly inhibited in a medium containing IL-1β at a concentration of 2000 pg/ml (PTH in % of control: 63.5 ± 5.3), n=10 (p<0.01). The inhibitory effect of IL-1β was not found in preparations of dispersed cells. The inhibitory effect of IL-1β could be counteracted by the IL-1 receptor antagonist (IL-1ra), indicating that the inhibitory effect was mediated through the specific IL-1 receptor on the parathyroid cells. IL-1β (2000 pg/ml) up-regulated CaRmRNA levels to 180% of control, whereas no change in PTHmRNA was found. IL-1ra abolished the upregulating effect of IL-1β on the CaRmRNA. This study demonstrates a direct effectin vitroof IL-1β on PTH secretion from bovine parathyroid glands, an effect which may be mediated at least in part through the specific IL-1 receptor causing an upregulation of the calcium-sensing receptor mRNA. IL-1 might therefore play a role as a auto- and/or paracrine factor in the regulation of the PTH secretion.     

The calcium-sensing receptor in taste tissue

Calcium is an essential nutrient that induces a distinctive taste quality, but the sensing mechanism of calcium in the tongue is poorly understood. A recent study linked calcium to T1R3 receptor. Here, we propose another system for calcium taste involving the extracellular calcium-sensing receptor (CaSR). This G protein-coupled receptor that responds to calcium and magnesium cations is involved in calcium homeostasis regulating parathyroid and kidney functions. In this study, CaSR was found in isolated taste buds from rats and mice. It was expressed in a subset of cells in circumvallate and foliate papillae, with fewer cells in the fungiform papillae. This is the first evidence in mammals that locates CaSR in gustatory tissue and provides the basis for better understanding not only calcium taste but also the taste of multiple CaSR agonists.     

Cryptorchidism: an indicator of testicular dysgenesis?

Cryptorchidism is a common ailment of new-born boys, affecting 1–9% of full term boys at birth. Cryptorchidism has been associated with an increased risk of testicular cancer and reduced fertility. Aetiology of cryptorchidism remains obscure in most cases. Familial occurrence suggests a heritable susceptibility to cryptorchidism; however, seasonal variation in the incidence of cryptorchidism suggests that environmental factors also contribute. Testicular descent is characterised by androgen-dependent regression of cranial suspensory ligament and androgen + insulin-like hormone 3 (Insl3)-dependent gubernacular outgrowth. Even though hormonal defects are rarely detected in patients, both hypo-and hypergonadotropic hormonal patterns have been associated with cryptorchidism. Moreover, cryptorchid boys have significantly reduced serum androgen bioactivity at 3 months of age when normal boys have a strong surge of reproductive hormones. Defects in Insl3 action cause cryptorchidism in male mice, and over-expression in female mice causes ovarian descent. Defects in leucine-rich repeat-containing G-protein-coupled receptor 8/G-protein-coupled receptor affecting testis descent (LGR8/GREAT), the receptor for Insl3, manifest the same phenotype as Insl3 knockout mutants. Even though mutations found in Insl3 and LGR8/GREAT genes are not a common cause of cryptorchidism in patients, it remains to be resolved whether low Insl3 levels during development are associated with cryptorchidism. Cryptorchidism may reflect foetal testicular dysgenesis that may later manifest as subfertility or testicular cancer.This work was supported by the Turku University Central Hospital, the Academy of Finland and the European Commission (contracts BMH4-CT96-0314, QLK4-CT1999-01422, QLK4-CT2001-00269 and QLK4-CT2002-00603).     

Aging influences the cardiac macrophage phenotype and function during steady state and during inflammation

Aging‐mediated immune dysregulation affects the normal cardiac immune cell phenotypes and functions, resulting in cardiac distress. During cardiac inflammation, immune activation is critical for mounting the regenerative responses to maintain normal heart function. We investigated the impact of aging on myeloid cell phenotype and function during cardiac inflammation induced by a sub‐lethal dose of LPS. Our data show that hearts of old mice contain more myeloid cells than the hearts of young mice. However, while the number of monocytic‐derived suppressor cells did not differ between young and old mice, monocytic‐derived suppressor cells from old mice were less able to suppress T‐cell proliferation. Since cardiac resident macrophages (CRMs) are important for immune surveillance, clearance of dead cells, and tissue repair, we focused our studies on CRMs phenotype and function during steady state and LPS treatment. In the steady state, we observed significantly more MHC‐II^low and MHC‐II^high CRMs in the hearts of old mice; however, these populations were decreased in both young and aged mice upon LPS treatment and the decrease in CRM populations correlated with defects in cardiac electrical activity. Notably, mice treated with a liver X receptor (LXR) agonist showed an increase in MerTK expression in CRMs of both young and old mice, which resulted in the reversal of cardiac electrical dysfunction caused by lipopolysaccharide (LPS). We conclude that aging alters the phenotype of CRMs, which contributes to the dysregulation of cardiac electrical dysfunction during infection in aged mice.     

The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH

Patients with neonatal severe hyperparathyroidism (NSHPT) are homozygous for the calcium-sensing receptor (CaR) mutation and have very high circulating PTH, abundant parathyroid hyperplasia, and severe life-threatening hypercalcemia. Mice with homozygous deletion of CaR mimic the syndrome of NSHPT. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)(2)D(3) or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR-deficient (CaR(-/-)) mice to those of double homozygous CaR- and 1α(OH)ase-deficient [CaR(-/-)1α(OH)ase(-/-)] mice or those of double homozygous CaR- and PTH-deficient [CaR(-/-)PTH(-/-)] mice at 2 weeks of age. Compared to wild-type littermates, CaR(-/-) mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, and severe skeletal growth retardation. Chondrocyte proliferation and PTHrP expression in growth plates were reduced significantly, whereas trabecular volume, osteoblast number, osteocalcin-positive areas, expression of the ALP, type I collagen, osteocalcin genes, and serum ALP levels were increased significantly. Deletion of 1α(OH)ase in CaR(-/-) mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, slight improvement in skeletal growth with increased chondrocyte proliferation and PTHrP expression, and further increases in indices of osteoblastic bone formation. Deletion of PTH in CaR(-/-) mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, undetectable serum PTH, normalization in skeletal growth with normal chondrocyte proliferation and enhanced PTHrP expression, and dramatic decreases in indices of osteoblastic bone formation. Our results indicate that reductions in hypercalcemia play a critical role in preventing the early lethality of CaR(-/-) mice and that defects in endochondral bone formation in CaR(-/-) mice result from effects of the marked elevation in serum calcium concentration and the decreases in serum phosphorus concentration and skeletal PTHrP levels, whereas the increased osteoblastic bone formation results from direct effects of PTH.