Stacy Pratt

EXPRESSION AND SIGNAL TRANSDUCTION OF CALCIUM-SENSING RECEPTORS IN CARTILAGE AND BONE

We previously showed that Ca 21 -sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca 21 ] ([Ca 21 ]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (;140 ‐190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca 21 ]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca 21 ]o reduced PTH-induced cAMP formation. Raising [Ca 21 ]o also increased intracellular free [Ca 21 ]i n SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca 21 ]o may couple to signaling pathways important in skeletal metabolism. (Endocrinology 140: 5883‐5893, 1999)

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...

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.