Tsui-Hua Chen

The Extracellular Calcium-Sensing Receptor (CaSR) Is a Critical Modulator of Skeletal Development

The extracellular calcium-sensing receptor (CaSR) is essential for embryonic and postnatal skeletal development. Bone Censor The extracellular calcium-sensing receptor (CaSR) is a guanine nucleotide-binding protein (G protein)–coupled receptor (GPCR) that responds to changes in the concentration of extracellular calcium ([Ca2+]e) and modulates various functions of parathyroid cells (PTCs), chondrocytes (the cells that produce cartilage), osteoblasts, and renal tubular cells. Previous attempts to characterize the CaSR in Casr−/− mice have been hampered by the expression of an alternatively spliced form of the receptor that provides at least partial compensation for loss of the full-length receptor (see the Perspective by Brown and Lian). Chang et al. have now developed mice with cell type–specific knockout of Casr that do not express the alternative receptor. Mice with PTC- or osteoblast-specific deletion of Casr were viable but had postnatal skeletal defects. Unexpectedly, knockout of Casr in chondrocytes was lethal. Mice in which chondrocyte-specific knockout of Casr was induced late in embryonic life were viable but had delayed growth plate development. Together, these findings reveal a previously unappreciated role for the CaSR in embryonic and postnatal skeletal development. The extracellular Ca2+-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.

The N-terminal Region of the Third Intracellular Loop of the Parathyroid Hormone (PTH)/PTH-related Peptide Receptor Is Critical for Coupling to cAMP and Inositol Phosphate/Ca2+ Signal Transduction Pathways

Structural determinants within the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor that mediate G-protein activation of adenylate cyclase and phospholipase C are unknown. We investigated the role of the N-terminal region of the third intracellular loop of the opossum PTH/PTHrP receptor in coupling to two signal transduction pathways. We mutated residues in this region by tandem-alanine scanning and expressed these mutant receptors in COS-7 cells and/or Xenopus oocytes. All mutant receptors retained high affinity PTH binding in COS-7 cells, indistinguishable from wild-type receptors. Receptors with tandem-alanine substitutions in two N-terminal segments (377RVL379 and 381TKLR384) demonstrated impaired adenylate cyclase and phospholipase C activation. Receptor mutants with single-alanine substitutions scanning these two segments showed three different signaling defects in COS-7 cells. 1) Two mutant receptors (V378A and L379A) had reduced inositol phosphate (IP), but normal cAMP responses to PTH. 2) Mutant receptor T381A showed reduced cAMP, but wild-type IP responses to PTH. 3) Mutant receptor K382A demonstrated both markedly reduced cAMP and IP production due to PTH. In oocytes, mutants T381A and K382A showed decreased PTH-stimulated cAMP accumulation and intracellular Ca2+ mobilization. Thus, the N-terminal region of the third intracellular loop of this receptor plays a critical role in coupling to both Gs- and Gq-mediated second-messenger generation.

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera.

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell‐surface Ca2+‐sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca2+, 5.5 mM Mg2+, or 10 μM Gd3+ significantly increased45Ca efflux (p < 0.01). InsP accumulation also increased dramatically when adding these cations to human embryonic kidney (HEK) 293 cells stably transfected with wild‐type bovine parathyroid CaR cDNA. Raising the extracellular [Ca2+] ([Ca2+]o) from 0.1 to >1.4 mM in oocytes and to >1.0 mM in HEK 293 cells stimulated significant increments in45Ca efflux and InsP accumulation, respectively (p < 0.05). In contrast, Ca2+ and Mg2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA. In HEK 293 cells stably expressing CaR cDNA, there were significant reductions in cAMP content when adding high Ca2+, Mg2+, Gd3+, or the CaR modulator NPS R‐467. Three region‐specific anti‐CaR peptide antisera immunoblotted bands of ∼140 and 155 kDa in membranes from CaR‐transfected HEK 293 cells and bovine parathyroid tissue. Immunocytochemistry demonstrated strong cell‐surface staining in CaR‐transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These results indicate that the activation of the recombinant CaR by extracellular Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+]o.

Extracellular Ca2+-Sensing Receptors Modulate Matrix Production and Mineralization in Chondrogenic RCJ3.1C5.18 Cells

Previous studies in chondrogenic RCJ3.1C5.18 (C5.18) cells showed that growth of these cells at high extracellular Ca2+ concentrations ([Ca2+]o) reduced the expression of markers of early chondrocyte differentiation. These studies addressed whether raising [Ca2+]o accelerates C5.18 cell differentiation and whether Ca2+ receptors (CaRs) are involved in coupling changes in [Ca2+]o to cellular responses. We found that high [Ca2+]o increased expression of osteopontin (OP), osteonectin, and osteocalcin, all markers of terminal differentiation, in C5.18 cells and increased the production of matrix mineral. Overexpression of wild-type CaR cDNA in C5.18 cells suppressed proteoglycan synthesis and aggrecan RNA, two early differentiation markers, and increased OP expression. The sensitivity of these parameters to changes in [Ca2+]o was significantly increased, as indicated by left-shifted dose-responses. In contrast, stable expression of a signaling-defective CaR mutant (Phe707Trp CaR) in C5.18 cells, presumably th...

Complex Formation with the Type B γ-Aminobutyric Acid Receptor Affects the Expression and Signal Transduction of the Extracellular Calcium-sensing Receptor STUDIES WITH HEK-293 CELLS AND NEURONS

We co-immunoprecipitated the Ca2+-sensing receptor (CaR) and type B γ-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 [Ca2+] ([Ca2+]e). In contrast, coexpressing CaRs and GABA-B-R2s enhanced CaR expression and signaling responses to raising [Ca2+]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 Ca2+-dependent receptor activation in target tissues.

Osteoblast extracellular Ca2+-sensing receptor regulates bone development, mineralization, and turnover

The extracellular Ca2+‐sensing receptor (CaR), a G protein‐coupled receptor responsible for maintenance of calcium homeostasis, is implicated in regulation of skeletal metabolism. To discern the role of the osteoblast CaR in regulation of bone development and remodeling, we generated mice in which the CaR is excised in a broad population of osteoblasts expressing the 3.6‐kb a1(I) collagen promoter. Conditional knockouts had abnormal skeletal histology at birth and developed progressively reduced mineralization secondary to retarded osteoblast differentiation, evident by significantly reduced numbers of osteoblasts and decreased expression of collagen I, osteocalcin, and sclerostin mRNAs. Elevated expression of ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1, and osteopontin mRNAs in the conditional knockout indicate altered regulation of genes important in mineralization. Knockout of the osteoblast CaR also resulted in increased expression of the receptor activator of NF‐κB ligand (RANKL), the major stimulator of osteoclast differentiation and function, consistent with elevated osteoclast numbers in vivo. Osteoblasts from the conditional knockouts exhibited delayed differentiation, reduced mineralizing capacity, altered expression of regulators of mineralization, and increased ability to promote osteoclastogenesis in coculture experiments. We conclude that CaR signaling in a broad population of osteoblasts is essential for bone development and remodeling and plays an important role in the regulation of differentiation and expression of regulators of bone resorption and mineralization.

Sex and age modify biochemical and skeletal manifestations of chronic hyperparathyroidism by altering target organ responses to Ca2+ and parathyroid hormone in mice.

We studied mice with or without heterozygous deletion of the Casr in the parathyroid gland (PTG) [PTGCaSR(+/–)] to delineate effects of age and sex on manifestations of hyperparathyroidism (HPT). In control mice, aging induced a left‐shift in the Ca2+/parathyroid hormone (PTH) set point accompanied by increased PTG CaSR expression along with lowered serum Ca2+ and mildly increased PTH levels, suggesting adaptive responses of PTGs to aging‐induced changes in mineral homeostasis. The aging effects on Ca2+/PTH set point and CaSR expression were significantly blunted in PTGCaSR(+/–) mice, who showed instead progressively elevated PTH levels with age, especially in 12‐month‐old females. These 12‐month‐old knockout mice demonstrated resistance to their high PTH levels in that serum 1,25‐dihydroxyvitamin D (1,25‐D) levels and RNA expression of renal Cyp27b1 and expression of genes involved in Ca2+ transport in kidney and intestine were unresponsive to the rising PTH levels. Such changes may promote negative Ca2+ balance, which further exacerbate the HPT. Skeletal responses to HPT were age‐, sex‐, and site‐dependent. In control mice of either sex, trabecular bone in the distal femur decreased whereas cortical bone in the tibiofibular junction increased with age. In male PTGCaSR(+/–) mice, anabolic actions of the elevated PTH levels seemed to protect against trabecular bone loss at ≥3 months of age at the expense of cortical bone loss. In contrast, HPT produced catabolic effects on trabecular bone and anabolic effects on cortical bone in 3‐month‐old females; but these effects reversed by 12 months, preserving trabecular bone in aging mice. We demonstrate that the CaSR plays a central role in the adaptive responses of parathyroid function to age‐induced changes in mineral metabolism and in target organ responses to calciotropic hormones. Restraining the ability of the PTG to upregulate CaSRs by heterozygous gene deletion contributes to biochemical and skeletal manifestations of HPT, especially in aging females.

Protein kinase C activation blocks calcium receptor signaling in Xenopus laevis oocytes.

We examined whether calcium receptor (CaR) signaling is affected by protein kinase C (PKC) activation by assessing the effects of phorbol-12-myristate-13-acetate (PMA) on 45Ca2+ efflux from Xenopus laevis oocytes expressing wild-type (WT) and mutant bovine parathyroid CaRs. Raising extracellular [Ca2+] ([Ca2+]0) from 0.5 to 5.5 mM increased 45Ca efflux (26 +/- 3-fold) in oocytes expressing full-length and C-terminally truncated receptor (amino acid 1-895). These increases in 45Ca efflux were blocked by 88 +/- 3% after PMA treatment for 20 min. Three consensus PKC phosphorylation sites (Thr-647, Ser-795, and Thr-889) were mutated in the context of the full-length and truncated CaR. PMA treatment inhibited high [Ca2+]0-induced responses in oocytes expressing the Ser795Ala CaR (1-895), Thr889Ala CaR (1-895), and Ser795Ala/Thr889Ala CaR (1-895) by 30-40% compared with untreated controls (P < 0.05). A triple mutant of the full-length CaR demonstrated similarly reduced susceptibility to inhibition of 45Ca efflux by PMA. Thus, these sites are important in mediating the effects of PKC activation on CaRs, but other residues and effector molecules are likely to participate in the effects of PKC on CaR-induced signal transduction in target cells.

Regulation of extracellular calcium-activated cation currents by cAMP in parathyroid cells

Parathyroid cells express Ca2+-sensing receptors that couple changes in the extracellular Ca2+ concentration ([Ca2+]o) to increases in the intracellular free Ca2+ concentration ([Ca2+]i) and to the suppression of parathyroid hormone secretion. Using whole cell patch clamping, we previously identified voltage-independent Ca2+-conducting currents in bovine parathyroid cells that increased with rising [Ca2+]o and were blocked by Cd2+ and nifedipine. Because cAMP-dependent phosphorylation regulates dihydropyridine-sensitive Ca2+ channels in other systems, we tested whether cAMP modulates these currents. At 0.7 mM Ca2+, nonselective Ca2+-conducting currents were suppressed by 30-50% when the recording pipette was perfused with cAMP. High-[Ca2+]o-induced increases in membrane currents were also abrogated. The effects of cAMP were reversible and dose dependent (3 x 10(-9) to 3 x 10(-3) M) and required ATP in the pipette solution. Perfusion of the cell interior with the catalytic subunit of protein kinase A mimicked the effects of cAMP, as did perfusion of the bath with the adenylate cyclase activator forskolin. These findings support the idea that cAMP-dependent phosphorylation suppresses high-[Ca2+]o-induced cation currents and may play a role in regulating ion fluxes in parathyroid cells.Parathyroid cells express Ca2+-sensing receptors that couple changes in the extracellular Ca2+ concentration ([Ca2+]o) to increases in the intracellular free Ca2+ concentration ([Ca2+]i) and to the suppression of parathyroid hormone secretion. Using whole cell patch clamping, we previously identified voltage-independent Ca2+-conducting currents in bovine parathyroid cells that increased with rising [Ca2+]oand were blocked by Cd2+ and nifedipine. Because cAMP-dependent phosphorylation regulates dihydropyridine-sensitive Ca2+channels in other systems, we tested whether cAMP modulates these currents. At 0.7 mM Ca2+, nonselective Ca2+-conducting currents were suppressed by 30-50% when the recording pipette was perfused with cAMP. High-[Ca2+]o-induced increases in membrane currents were also abrogated. The effects of cAMP were reversible and dose dependent (3 × 10-9 to 3 × 10-3 M) and required ATP in the pipette solution. Perfusion of the cell interior with the catalytic subunit of protein kinase A mimicked the effects of cAMP, as did perfusion of the bath with the adenylate cyclase activator forskolin. These findings support the idea that cAMP-dependent phosphorylation suppresses high-[Ca2+]o-induced cation currents and may play a role in regulating ion fluxes in parathyroid cells.