Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells.

BACKGROUND: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. METHODS: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox ). RESULTS: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. CONCLUSIONS: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis.

PTH hypersecretion triggered by a GABAB1 and Ca2+-sensing receptor heterocomplex in hyperparathyroidism.

Molecular mechanisms mediating tonic secretion of parathyroid hormone (PTH) in response to hypocalcaemia and hyperparathyroidism (HPT) are unclear. Here we demonstrate increased heterocomplex formation between the calcium-sensing receptor (CaSR) and metabotropic γ-aminobutyric acid (GABA) B1 receptor (GABAB1R) in hyperplastic parathyroid glands (PTGs) of patients with primary and secondary HPT. Targeted ablation of GABAB1R or glutamic acid decarboxylase 1 and 2 in PTGs produces hypocalcaemia and hypoparathyroidism, and prevents PTH hypersecretion in PTGs cultured from mouse models of hereditary HPT and dietary calcium-deficiency. Cobinding of the CaSR/GABAB1R complex by baclofen and high extracellular calcium blocks the coupling of heterotrimeric G-proteins to homomeric CaSRs in cultured cells and promotes PTH secretion in cultured mouse PTGs. These results combined with the ability of PTG to synthesize GABA support a critical autocrine action of GABA/GABAB1R in mediating tonic PTH secretion of PTGs and ascribe aberrant activities of CaSR/GABAB1R heteromer to HPT.

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice.

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation-an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca2+ ] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.

Phosphate acts directly on the calcium-sensing receptor to stimulate parathyroid hormone secretion.

Extracellular phosphate regulates its own renal excretion by eliciting concentration-dependent secretion of parathyroid hormone (PTH). However, the phosphate-sensing mechanism remains unknown and requires elucidation for understanding the aetiology of secondary hyperparathyroidism in chronic kidney disease (CKD). The calcium-sensing receptor (CaSR) is the main controller of PTH secretion and here we show that raising phosphate concentration within the pathophysiologic range for CKD significantly inhibits CaSR activity via non-competitive antagonism. Mutation of residue R62 in anion binding site-1 abolishes phosphate-induced inhibition of CaSR. Further, pathophysiologic phosphate concentrations elicit rapid and reversible increases in PTH secretion from freshly-isolated human parathyroid cells consistent with a receptor-mediated action. The same effect is seen in wild-type murine parathyroid glands, but not in CaSR knockout glands. By sensing moderate changes in extracellular phosphate concentration, the CaSR represents a phosphate sensor in the parathyroid gland, explaining the stimulatory effect of phosphate on PTH secretion.

Morpholino oligonucleotide knockdown of the extracellular calcium-sensing receptor impairs early skeletal development in zebrafish.

The complex vertebrate skeleton depends on regulated cell activities to lay down protein matrix and mineral components of bone. As a distinctive vertebrate characteristic, bone is a storage site for physiologically-important calcium ion. The extracellular calcium-sensing receptor (CaSR) is linked to homeostatic regulation of calcium through its expression in endocrine glands that secrete calcium homeostatic hormones, in Ca(2+)- and ion-transporting epithelia, and in skeleton. Since CaSR is restricted in its presence to the chordate-vertebrate evolutionary lineage, we propose there to be important functional ties between CaSRs and vertebrate skeleton in the context of that group's characteristic form of calcium-mineralized skeleton. Since little is known about CaSR in the skeletal biology of non-mammalian vertebrates, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry were applied to adult and embryonic zebrafish to reveal CaSR transcript and protein expression in several tissues, including, among these, chondrocytes and developing bone and notochord as components in skeletal development. Morpholino oligonucleotide (MO) knockdown technique was used to probe CaSR role(s) in the zebrafish model system. By RT-PCR assessment, injection of a splice-inhibiting CaSR MO reduced normally-spliced Casr gene transcript expression measured at 2days postfertilization (dpf). Corresponding to the knockdown of normally-spliced mRNA by the CaSR MO, we observed a morphant phenotype characterized by stunted growth and disorganization of the notochord and axial skeleton by 1dpf. We conclude that, like its critically important role in normal bone development in mammals, CaSR is essential in skeletogenesis in fishes.

Vertebrate extracellular calcium-sensing receptor evolution: selection in relation to life history and habitat.

Ionic calcium (Ca(2+)) supports essential functions within physiological systems, and consequently its concentration is homeostatically regulated within narrow bounds in the body fluids of animals through endocrine effects at ion-transporting osmoregulatory tissues. In vertebrates, extracellular Ca(2+) is detected at the cell surface by the extracellular calcium-sensing receptor (CaSR), a member of the G protein-coupled receptor (GPCR) superfamily. Interestingly, the taxonomic distribution of CaSRs is restricted to vertebrates, with some CaSR-like receptors apparently present in non-vertebrate chordates. Since bone is a known Ca(2+) storage site and is characteristically restricted to the vertebrate lineage, we hypothesized a functional association of CaSR with vertebrate skeleton that may have an ancient origin. Protein sequence alignment and phylogenetic analysis of vertebrate CaSRs and related GPCRs of the glutamate receptor-like family expose similarities and indel differences among these receptors, and reveal the evolutionary history of CaSRs. Evolutionary selection was tested statistically by evaluating the relationship between non-synonymous (replacement, dN) versus synonymous (silent, dS) amino acid substitution rates (as dN/dS) of protein-coding DNA sequences among branches of the estimated protein phylogeny. On a background of strong purifying selection (dN/dS<1) in the CaSR phylogeny, statistical evidence for adaptive evolution (dN/dS>1) was detected on some branches to major clades in the CaSR phylogeny, especially to the tetrapod vertebrate CaSRs and chordate CaSR-like branches. Testing also revealed overall purifying selection at the codon level. At some sites relaxation from strong purifying selection was seen, but evidence for adaptive evolution was not detected for individual sites. The results suggest purifying selection of CaSRs, and of adaptive evolution among some major vertebrate clades, reflecting clade specific differences in natural history and organismal biology, including skeletal involvement in calcium homeostasis.

Skeletal tissues in Mozambique tilapia (Oreochromis mossambicus) express the extracellular calcium-sensing receptor.

Molecular phylogenetic analysis suggests that the extracellular calcium-sensing receptor (CaSR) emerged evolutionarily in association with the chordate-vertebrate lineage. Our studies overall explore the evolution of CaSRs, and the possible historical linkage of CaSRs to vertebrate skeleton as functional components of calcium homeostasis through regulated storage and/or release. We applied both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) to evaluate Casr gene and CaSR protein expression, respectively, in skeletal tissues of a cichlid teleost, the Mozambique tilapia (Oreochromis mossambicus). By RT-PCR, CaSR gene (Casr) expression was observed in skull and vertebral column (including notochordal tissues). Relative to skeleton, IHC revealed CaSR protein expression in notochordal sheath cells within the vertebral canal, in scleroblasts associated with body surface scales, and in chondrocytes within hyaline cartilage. Although closely apposed cells border the acellular bone in tilapia, these cells were only weakly immunostained. We conclude, therefore, that CaSR is expressed in skeletal tissues of tilapia, an advanced teleost fish, and that Casr may be part of a genetic network associated with vertebrate skeletal system. Our immunohistochemical examination also newly revealed CaSR protein expression in epidermis and red muscle of fishes.