Calcium-sensing receptor deletion in the mouse esophagus alters barrier function.

Calcium-sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca2+ concentrations. CaSR has been reported to play a role in glandular and fluid secretion in the gastrointestinal tract and to regulate differentiation and proliferation of skin keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating keratin 5 CreER;CaSRFlox+/+compound mutants, in which loxP sites flank exon 7 of CaSR gene. Recombination was initiated with multiple tamoxifen injections, and we demonstrated exon 7 deletion by PCR analysis of genomic DNA. Quantitative real-time PCR and Western blot analyses showed a significant reduction in CaSR mRNA and protein expression in the knockout mice (EsoCaSR-/-) as compared with control mice. Microscopic examination of EsoCaSR-/- esophageal tissues showed morphological changes including elongation of the rete pegs, abnormal keratinization and stratification, and bacterial buildup on the luminal epithelial surface. Western analysis revealed a significant reduction in levels of adherens junction proteins E-cadherin and ß catenin and tight junction protein claudin-1, 4, and 5. Levels of small GTPase proteins Rac/Cdc42, involved in actin remodeling, were also reduced. Ussing chamber experiments showed a significantly lower transepithelial resistance in knockout (KO) tissues. In addition, luminal-to-serosal-fluorescein dextran (4 kDa) flux was higher in KO tissues. Our data indicate that CaSR plays a role in regulating keratinization and cell-cell junctional complexes and is therefore important for the maintenance of the barrier function of the esophagus.NEW & NOTEWORTHY The esophageal stratified squamous epithelium maintains its integrity by continuous proliferation and differentiation of the basal cells. Here, we demonstrate that deletion of the calcium-sensing receptor, a G protein-coupled receptor, from the basal cells disrupts the structure and barrier properties of the epithelium.

Spermine on Endothelial Extracellular Vesicles Mediates Smoking-Induced Pulmonary Hypertension Partially Through Calcium-Sensing Receptor.

Objective- This study aims to determine whether and how the enriched metabolites of endothelial extracellular vesicles (eEVs) are critical for cigarette smoke-induced direct injury of endothelial cells and the development of pulmonary hypertension, rarely explored in contrast to long-investigated mechanisms secondary to chronic hypoxemia. Approach and Results- Metabonomic screen of eEVs from cigarette-smoking human subjects reveals prominent elevation of spermine-a polyamine metabolite with potent agonist activity for the extracellular CaSR (calcium-sensing receptor). CaSR inhibition with the negative allosteric modulator Calhex231 or CaSR knockdown attenuates cigarette smoke-induced pulmonary hypertension in rats without emphysematous changes in lungs or chronic hypoxemia. Cigarette smoke exposure increases the generation of spermine-positive eEVs and their spermine content. Immunocytochemical staining and immunogold electron microscopy recognize the spermine enrichment not only within the cytosol but also on the outer surface of eEV membrane. The repression of spermine synthesis, the inhibitory analog of spermine, N1-dansyl-spermine, Calhex231, or CaSR knockdown profoundly suppresses eEV exposure-mobilized cytosolic calcium signaling, pulmonary artery constriction, and smooth muscle cell proliferation. Confocal imaging of immunohistochemical staining demonstrates the migration of spermine-positive eEVs from endothelium into smooth muscle cells in pulmonary arteries of cigarette smoke-exposed rats. The repression of spermine synthesis or CaSR knockout results in attenuated development of pulmonary hypertension induced by an intravascular administration of eEVs. Conclusions- Cigarette smoke enhances eEV generation with spermine enrichment at their outer surface and cytosol, which activates CaSR and subsequently causes smooth muscle cell constriction and proliferation, therefore, directly leading to the development of pulmonary hypertension.

The calcium-sensing receptor in bone metabolism: from bench to bedside and back.

UNLABELLED: The calcium-sensing receptor (CaSR), a key player in the maintenance of calcium homeostasis, can influence bone modeling and remodeling by directly acting on bone cells, as demonstrated by in vivo and in vitro evidence. The modulation of CaSR signaling can play a role in bone anabolism. INTRODUCTION: The calcium-sensing receptor (CaSR) is a key player in the maintenance of calcium homeostasis through the regulation of PTH secretion and calcium homeostasis, thus indirectly influencing bone metabolism. In addition to this role, in vitro and in vivo evidence points to direct effects of CaSR in bone modeling and remodeling. In addition, the activation of the CaSR is one of the anabolic mechanisms implicated in the action of strontium ranelate, to reduce fracture risk. METHODS: This review is based upon the acquisition of data from a PubMed enquiry using the terms "calcium sensing receptor," "CaSR" AND "bone remodeling," "bone modeling," "bone turnover," "osteoblast," "osteoclast," "osteocyte," "chondrocyte," "bone marrow," "calcilytics," "calcimimetics," "strontium," "osteoporosis," "skeletal homeostasis," and "bone metabolism." RESULTS: A fully functional CaSR is expressed in osteoblasts and osteoclasts, so that these cells are able to sense changes in the extracellular calcium and as a result modulate their behavior. CaSR agonists (calcimimetics) or antagonists (calcilytics) have the potential to indirectly influence skeletal homeostasis through the modulation of PTH secretion by the parathyroid glands. The bone anabolic effect of strontium ranelate, a divalent cation used as a treatment for postmenopausal and male osteoporosis, might be explained, at least in part, by the activation of CaSR in bone cells. CONCLUSIONS: Calcium released in the bone microenvironment during remodeling is a major factor in regulating bone cells. Osteoblast and osteoclast proliferation, differentiation, and apoptosis are influenced by local extracellular calcium concentration. Thus, the calcium-sensing properties of skeletal cells can be exploited in order to modulate bone turnover and can explain the bone anabolic effects of agents developed and employed to revert osteoporosis.

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.

Induction of CaSR expression circumvents the molecular features of malignant CaSR null colon cancer cells.

We recently reported on the isolation and characterization of calcium sensing receptor (CaSR) null human colon cancer cells (Singh et al., Int J Cancer 2013; 132: 1996-2005). CaSR null cells possess a myriad of molecular features that are linked to a highly malignant and drug resistant phenotype of colon cancer. The CaSR null phenotype can be maintained in defined human embryonic stem cell culture medium. We now show that the CaSR null cells can be induced to differentiate in conventional culture medium, regained the expression of CaSR with a concurrent reversal of the cellular and molecular features associated with the null phenotype. These features include cellular morphology, expression of colon cancer stem cell markers, expression of survivin and thymidylate synthase and sensitivity to fluorouracil. Other features include the expression of epithelial mesenchymal transition linked molecules and transcription factors, oncogenic miRNAs and tumor suppressive molecule and miRNA. With the exception of cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, is directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. We further report that methylation and demethylation of the CaSR gene promoter underlie CaSR expression. Due to the malignant nature of the CaSR null cells, inclusion of the CaSR null phenotype in disease management may improve on the mortality of this disease. Because CaSR is a robust promoter of differentiation and mediates its action through diverse mechanisms and pathways, inactivation of CaSR may serve as a new paradigm in colon carcinogenesis.

Alterations in phosphorus, calcium and PTHrP contribute to defects in dental and dental alveolar bone formation in calcium-sensing receptor-deficient mice.

To determine whether the calcium-sensing receptor (CaR) participates in tooth formation and dental alveolar bone development in mandibles in vivo, we examined these processes, as well as mineralization, in 2-week-old CaR-knockout (CaR(-/-)) mice. We also attempted to rescue the phenotype of CaR(-/-) mice by genetic means, in mice doubly homozygous for CaR and 25-hydroxyvitamin D 1alpha-hydroxylase [1alpha(OH)ase] or parathyroid hormone (Pth). In CaR(-/-) mice, which exhibited hypercalcemia, hypophosphatemia and increased serum PTH, the volumes of teeth and of dental alveolar bone were decreased dramatically, whereas the ratio of the area of predentin to total dentin and the number and surface of osteoblasts in dental alveolar bone were increased significantly, as compared with wild-type littermates. The normocalcemia present in CaR(-/-);1alpha(OH)ase(-/-) mice only slightly improved the defects in dental and alveolar bone formation observed in the hypercalcemic CaR(-/-) mice. However, these defects were completely rescued by the additional elimination of hypophosphatemia and by an increase in parathyroid hormone-related protein (PTHrP) expression in the apical pulp, Hertwig's epithelial root sheath and mandibular tissue in CaR(-/-); Pth(-/-) mice. Therefore, alterations in calcium, phosphorus and PTHrP contribute to defects in the formation of teeth and alveolar bone in CaR-deficient mice. This study indicates that CaR participates in the formation of teeth and in the development of dental alveolar bone in mandibles in vivo, although it appears to do so largely indirectly.

Deficiency of the calcium-sensing receptor in the kidney causes parathyroid hormone-independent hypocalciuria.

Rare loss-of-function mutations in the calcium-sensing receptor (Casr) gene lead to decreased urinary calcium excretion in the context of parathyroid hormone (PTH)-dependent hypercalcemia, but the role of Casr in the kidney is unknown. Using animals expressing Cre recombinase driven by the Six2 promoter, we generated mice that appeared grossly normal but had undetectable levels of Casr mRNA and protein in the kidney. Baseline serum calcium, phosphorus, magnesium, and PTH levels were similar to control mice. When challenged with dietary calcium supplementation, however, these mice had significantly lower urinary calcium excretion than controls (urinary calcium to creatinine, 0.31±0.03 versus 0.63±0.14; P=0.001). Western blot analysis on whole-kidney lysates suggested an approximately four-fold increase in activated Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). In addition, experimental animals exhibited significant downregulation of Claudin14, a negative regulator of paracellular cation permeability in the thick ascending limb, and small but significant upregulation of Claudin16, a positive regulator of paracellular cation permeability. Taken together, these data suggest that renal Casr regulates calcium reabsorption in the thick ascending limb, independent of any change in PTH, by increasing the lumen-positive driving force for paracellular Ca(2+) transport.

Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.

During mammalian ontogeny, haematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, where haematopoiesis occurs throughout adulthood. Unique features of bone that contribute to a microenvironmental niche for stem cells might include the known high concentration of calcium ions at the HSC-enriched endosteal surface. Cells respond to extracellular ionic calcium concentrations through the seven-transmembrane-spanning calcium-sensing receptor (CaR), which we identified as being expressed on HSCs. Here we show that, through the CaR, the simple ionic mineral content of the niche may dictate the preferential localization of adult mammalian haematopoiesis in bone. Antenatal mice deficient in CaR had primitive haematopoietic cells in the circulation and spleen, whereas few were found in bone marrow. CaR-/- HSCs from fetal liver were normal in number, in proliferative and differentiative function, and in migration and homing to the bone marrow. Yet they were highly defective in localizing anatomically to the endosteal niche, behaviour that correlated with defective adhesion to the extracellular matrix protein, collagen I. CaR has a function in retaining HSCs in close physical proximity to the endosteal surface and the regulatory niche components associated with it.

Adipocyte calcium sensing receptor is not involved in visceral adipose tissue inflammation or atherosclerosis development in hyperlipidemic Apoe-/- mice.

The calcium sensing receptor (CaSR) is a G-protein coupled receptor that especially plays an important role in the sensing of extracellular calcium to maintain its homeostasis. Several in-vitro studies demonstrated that CaSR plays a role in adipose tissue metabolism and inflammation, resulting in systemic inflammation and contributing to atherosclerosis development. The aim of this study was to investigate whether adipocyte CaSR plays a role in adipose tissue inflammation in-vivo and atherosclerosis development. By using a newly established conditional mature adipocyte specific CaSR deficient mouse on a hyperlipidemic and atherosclerosis prone Apoe-/- background it could be shown that CaSR deficiency in adipocytes does neither contribute to initiation nor to progression of atherosclerotic plaques as judged by the unchanged lesion size or composition. Additionally, CaSR deficiency did not influence gonadal visceral adipose tissue (vAT) inflammation in-vivo, although a small decrease in gonadal visceral adipose cholesterol content could be observed. In conclusion, adipocyte CaSR seems not to be involved in vAT inflammation in-vivo and does not influence atherosclerosis development in hyperlipidemic Apoe-/- mice.

Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis.

Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1ß release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1ß release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.

The full-length calcium-sensing receptor dampens the calcemic response to 1alpha,25(OH)2 vitamin D3 in vivo independently of parathyroid hormone.

1Alpha,25(OH)(2) vitamin D(3) [1,25(OH)(2)D(3)] increases serum Ca(2+) concentration in vivo, an action counteracted by activation of the Ca(2+)-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca(2+) excretion. Relatively little is known of the role the CaSR plays in this response through its potentially direct actions in kidney, gut, and bone independently of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)(2)D(3) in mice with targeted disruption of both the CaSR and PTH genes (C(-)P(-)) compared with that in mice with disruption of the PTH gene alone (C(+)P(-)) or wild-type mice (C(+)P(+)). After intraperitoneal injection of 0.5 ng/g body wt 1,25(OH)(2)D(3), peak calcemic responses were observed at 24 h in all three genotypes in association with 1) a greater increase in serum Ca(2+) in C(-)P(-) mice than in the other genotypes on a Ca(2+)-replete diet that was attenuated by a Ca(2+)-deficient diet and pamidronate, 2) increased urinary Ca(2+)-to-creatinine ratios (UCa/Cr) in the C(+)P(-) and C(+)P(+) mice but a lowered ratio in the C(-)P(-) mice on a Ca(2+)-replete diet, and 3) no increase in calcitonin (CT) secretion in the C(+)P(+) and C(+)P(-) mice and a small increase in the C(-)P(-) mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca(2+) transport at baseline in the duodenum and kidney, and injection of 1,25(OH)(2)D(3) increased gene expression 8 h later. However, the changes in the genes evaluated did not fully explain the differences in serum Ca(2+) seen among the genotypes. In conclusion, mice lacking the full-length CaSR have increased sensitivity to the calcemic action of 1,25(OH)(2)D(3) in the setting of PTH deficiency. This is principally from enhanced 1,25(OH)(2)D(3)-mediated gut Ca(2+) absorption and decreased renal Ca(2+) excretion, without any differences in bone-related release of Ca(2+) or CT secretion among the three genotypes that could explain the differences in their calcemic responses.

Calcimimetic acts on enteric neuronal CaSR to reverse cholera toxin-induced intestinal electrolyte secretion.

Treatment of acute secretory diarrheal illnesses remains a global challenge. Enterotoxins produce secretion through direct epithelial action and indirectly by activating enteric nervous system (ENS). Using a microperfused colonic crypt technique, we have previously shown that R568, a calcimimetic that activates the calcium-sensing receptor (CaSR), can act on intestinal epithelium and reverse cholera toxin-induced fluid secretion. In the present study, using the Ussing chamber technique in conjunction with a tissue-specific knockout approach, we show that the effects of cholera toxin and CaSR agonists on electrolyte secretion by the intestine can also be attributed to opposing actions of the toxin and CaSR on the activity of the ENS. Our results suggest that targeting intestinal CaSR might represent a previously undescribed new approach for treating secretory diarrheal diseases and other conditions with ENS over-activation.

Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium-Sensing Receptor.

BACKGROUND: Monocrotaline has been widely used to establish an animal model of pulmonary hypertension. The molecular target underlying monocrotaline-induced pulmonary artery endothelial injury and pulmonary hypertension remains unknown. The extracellular calcium-sensing receptor (CaSR) and particularly its extracellular domain hold the potential structural basis for monocrotaline to bind. This study aimed to reveal whether monocrotaline induces pulmonary hypertension by targeting the CaSR. METHODS AND RESULTS: Nuclear magnetic resonance screening through WaterLOGSY (water ligand-observed gradient spectroscopy) and saturation transfer difference on protein preparation demonstrated the binding of monocrotaline to the CaSR. Immunocytochemical staining showed colocalization of monocrotaline with the CaSR in cultured pulmonary artery endothelial cells. Cellular thermal shift assay further verified the binding of monocrotaline to the CaSR in pulmonary arteries from monocrotaline-injected rats. Monocrotaline enhanced the assembly of CaSR, triggered the mobilization of calcium signaling, and damaged pulmonary artery endothelial cells in a CaSR-dependent manner. Finally, monocrotaline-induced pulmonary hypertension in rats was significantly attenuated or abolished by the inhibitor, the general or lung knockdown or knockout of CaSR. CONCLUSIONS: Monocrotaline aggregates on and activates the CaSR of pulmonary artery endothelial cells to trigger endothelial damage and, ultimately, induces pulmonary hypertension.

The calcium-sensing receptor regulates PTHrP production and calcium transport in the lactating mammary gland.

Lactating mammals must supply large amounts of calcium to the mammary gland where it is transported across mammary epithelial cells and into milk. This demand for calcium is associated with transient loss of bone mass, triggered, in part, by the secretion of parathyroid hormone-related protein (PTHrP) from the mammary gland into the circulation. The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium ions. It is responsible for coordinating calcium homeostasis by regulating parathyroid hormone secretion in the parathyroid glands and by regulating calcium handling in the renal tubules. Previous studies had shown that the CaR is expressed on mammary epithelial cells during lactation, and it had been suggested that CaR signaling in the mammary gland helps to coordinate its production of PTHrP and calcium transport into milk. In this study, we examined mammary gland PTHrP production and calcium transport in CaR(+/-) mice, a genetic model of CaR insufficiency. We found that haploinsufficiency for the CaR resulted in increased PTHrP production both in vivo and in vitro. In contrast, CaR haploinsufficiency impaired calcium transport into milk in vivo and transepithelial calcium transport by mammary epithelial cells in vitro. These data provide genetic confirmation that the CaR regulates PTHrP production and calcium transport in the lactating mammary gland. This allows the mammary gland to become a calcium-sensing organ and to participate in systemic calcium homeostasis during lactation.

Hypocalciuric hypercalcemia presenting as neonatal rib fractures: a newly described mutation of the calcium-sensing receptor gene.

A 2-week-old infant presented with bilateral rib fractures, hypercalcemia, and subperiosteal bone erosions. Parathyroid hormone levels were elevated and urine calcium low. Her parent's laboratory test results were normal. Gene sequencing revealed a new mutation of the calcium-sensing receptor gene, causing severe neonatal hyperparathyroidism, a variant of hypocalciuric hypercalcemia. This is a rare cause of neonatal hyperparathyroidism and nonabusive fractures.

Expression and functional assessment of an alternatively spliced extracellular Ca2+-sensing receptor in growth plate chondrocytes.

The extracellular Ca(2+)-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR(-/-)) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [(Exon5(-))CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR(-/-) mice express (Exon5(-))CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of (Exon5(-))CaR in growth plates from CaR(-/-) mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human (Exon5(-))CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human (Exon5(-))CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR(-/-) GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR(-/-) mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably (Exon5(-))CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR(-/-) mice, high extracellular [Ca(2+)] ([Ca(2+)](e)) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca(2+)](e) also promoted the differentiation of CaR(-/-) GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of (Exon5(-))CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca(2+)](e) in GPCs in CaR(-/-) mice.

Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo.

BACKGROUND: The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium. To demonstrate regulatory effects of CaSR on calcitonin release, we studied calcitonin responsiveness to calcium in normal and CaSR heterozygous-ablated (Casr+/-) mice. Casr+/- mice have hypercalcemia and hypocalciuria, and live normal life spans. Each mouse received either 500 microl of normal saline or one of two doses of elemental calcium (500 micromol/kg or 5 mmol/kg) by intraperitoneal injection. Ionized calcium was measured at baseline and 10 minutes, and serum calcitonin was measured on the 10 minute sample. RESULTS: At baseline, Casr+/- mice had a higher blood calcium, and in response to the two doses of elemental calcium, had greater increments and peak levels of ionized calcium than their wild type littermates. Despite significantly higher ionized calcium levels, the calcitonin levels of Casr+/- mice were consistently lower than wild type at any ionized calcium level, indicating that the dose-response curve of calcitonin to increases in ionized calcium had been significantly blunted or shifted to the right in Casr+/- mice. CONCLUSIONS: These results confirm that the CaSR is a physiological regulator of calcitonin; therefore, in response to increases in ionized calcium, the CaSR inhibits parathyroid hormone secretion and stimulates calcitonin secretion.

Epithelial CaSR deficiency alters intestinal integrity and promotes proinflammatory immune responses.

The intestinal epithelium is equipped with sensing receptor mechanisms that interact with luminal microorganisms and nutrients to regulate barrier function and gut immune responses, thereby maintaining intestinal homeostasis. Herein, we clarify the role of the extracellular calcium-sensing receptor (CaSR) using intestinal epithelium-specific Casr(-/-) mice. Epithelial CaSR deficiency diminished intestinal barrier function, altered microbiota composition, and skewed immune responses towards proinflammatory. Consequently, Casr(-/-) mice were significantly more prone to chemically induced intestinal inflammation resulting in colitis. Accordingly, CaSR represents a potential therapeutic target for autoinflammatory disorders, including inflammatory bowel diseases.

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.