- Email: [email protected]
Calcimimetic and calcilytic drugs
S22
Abstracts
that OS-9 may have effects on CaSR processing and expression that could profoundly influence CaSR-related disease.
doi:10.1016/j.bone.2012.08.071
O53 Calcimimetic and calcilytic drugs E.F. Nemeth MetisMedica, Toronto, Canada Abstract: Drugs acting on the calcium receptor (CaR) can rapidly alter circulating levels of parathyroid hormone (PTH) and are therefore useful in the treatment of disorders of mineral metabolism. The type II calcimimetic cinacalcet is approved for treating secondary hyperparathyroidism (HPT) in patients receiving renal replacement therapy or in those with parathyroid carcinoma and is gaining approval for treating primary HPT in a number of EU countries. The type I calcimimetic KAI-4169 is currently in late stage clinical development for treating secondary HPT in patients with chronic kidney disease, so this agonist of the CaR might join the positive allosteric modulator cinacalcet as an effective treatment for HPT. The development of most calcilytics as therapies for osteoporosis has been discontinued and it is uncertain if the failure of calcilytics to generally increase bone mineral density is due to the pharmacodynamic or pharmacokinetic properties of these compounds. In contrast to type II calcimimetics, calcilytics do not show tissue selectivity and could neutralize anabolic effects of PTH by blocking skeletal CaRs. The amino alcohol chemotype calcilytics have therefore been repositioned for new indications: ronacaleret for stem cell transplantation and NPSP790 and NPSP795 for autosomal dominant hypocalcemia. The rationale for the latter indication derives not only from the ability of calcilytics to increase PTH secretion but additionally from their ability to inhibit renal excretion of calcium. New indications for calcimimetics and calcilytics will require the discovery of compounds that preferentially act on CaRs in tissues other than the parathyroid gland and kidney.
doi:10.1016/j.bone.2012.08.072
O54 The intimate link between calcium sensing receptor trafficking and signaling M.P. Grant, A. Stepanchick, A. Cavanaugh, G.E. Breitwieser Geisinger Clinic, Danville, PA, USA Abstract: Calcium sensing receptors (CaSR) regulate systemic calcium homeostasis at the parathyroid, kidney, intestine and bone, translating fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. CaSR, a family C G protein-coupled receptor (GPCR), operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. We recently defined a unique mechanism, agonist-driven insertional signaling (ADIS), which couples plasma membrane CaSR signaling to increased anterograde trafficking of CaSR, leading to a net increase in plasma membrane CaSR during agonist exposure. Signaling is also coupled to enhanced CaSR biosynthesis, allowing maintenance of intracellular reservoirs of mobilizable receptor. We used multi-wavelength total internal reflection fluorescence (TIRF) microscopy alternating with wild-field imaging to monitor intracellular calcium. Plasma membrane CaSR undergoes constitutive endocytosis without significant recycling, thus signaling is determined by the rate of insertion of CaSR at the plasma membrane. Sustained CaSR signaling results from agonist-driven, brefeldin A-sensitive trafficking of CaSR through the secretory pathway. The intracellular reservoir of mobilizable CaSR is depleted when glycosylation of newly synthesized receptors is blocked by tunicamycin, demonstrating the coupling of receptor biosynthesis to signaling. The continuous, signaling-dependent insertion of CaSR at the plasma membrane ensures a rapid response to alterations in extracellular calcium (or allosteric agonist) concentrations despite ongoing desensitization and endocytosis. Signaling-regulated CaSR plasma membrane abundance represents a unique adaptation of the normal mechanisms regulating GPCR signaling. Supported by Geisinger Clinic and NIH.
doi:10.1016/j.bone.2012.08.073
O55 Regulation of signalling pathways by extracellular calcium in colon cancer cells S. Tennakoona, B. Marianb, J. Schmidc, E. Kállaya a Department of Pathophysiology and Allergy Research,
Medical University of Vienna, Austria Department of Internal Medicine I, Institute of Cancer Research, Medical University of Vienna, Austria c Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria b
Abstract: The calcium sensing receptor (CaSR) is a G-protein coupled receptor, which is activated by a number of ligands stimulating various downstream cell signalling pathways. In addition to its main role in regulation of calcium homeostasis, CaSR is also involved in numerous other biological processes such as cell proliferation, differentiation and lipid membrane metabolism. Previous studies demonstrated that calcium regulates cell proliferation in colonocytes but the pathways or factors involved in this process are still need to be elucidated. The aim of this study is to investigate the pathways regulated by the activated CaSR in colonocytes. In HEK cells it seems that calcium activates the MAP/ERK (mitogen-activated protein kinase/extracellular-signal-regulated kinases) and the NFkappaB (Nuclear FactorKappaB) pathways through the CaSR. Therefore we studied the impact of calcium (2 mM) on these two proliferative pathways using luciferase reporter assay and western blot in the human colon cancer cell line Caco-2. Luciferase data showed that neither the MAP/ ERK nor the NFkappaB pathway is activated by treatment with calcium in colonocytes. Further, we have seen no changes in phospho-ERK expression after treatment with calcium for 10–40 min. However we observed a reduction of phospho-ERK expression after 8 and 24 h treatment with calcium. In conclusion, our data shows that in Caco-2 cells calcium does not activate either the MAP/ERK or NFkappaB pathway and that long-term treatment may even lead to their inhibition. The next step would be to look at other candidate signalling pathways and to confirm the involvement of CaSR by modulating its expression.
doi:10.1016/j.bone.2012.08.074
O56 Modulation of cAMP and cAMP-sensitive signaling by the calcium-sensing receptor A.D. Conigravea, V.A. Avlania, A. Christopoulosb, H.-C. Muna a School of Molecular Bioscience, University of Sydney, NSW 2006, Australia b Monash Institute of Pharmaceutical Sciences, Parkville 3052 Victoria, Australia Abstract: The calcium-sensing receptor (CaSR) modulates cAMP metabolism in parathyroid cells and various other cell-types as part of the mechanism by which it provides acute control of hormone secretion. In addition, the CaSR modulates the number and differentiation state of cells in diverse tissues including osteoblasts in bone, keratinocytes in skin and intestinal epithelial cells via the activation and/or inhibition of signaling pathways upstream of key transcriptional regulators e.g., ERK1/ 2 and SRE. In recent studies, we have investigated the mechanisms by which extracellular Ca2 + and positive allosteric modulators (PAMs) of the CaSR including cinacalcet, L-Phe, and S-methylglutathione control intracellular cAMP levels as well as the impact of CaSR modulators on cAMP-dependent transcription via the transcription factor CREB. With respect to cAMP levels in CaSR-expressing HEK-293 cells and parathyroid cells, the CaSR acts primarily to suppress cAMP synthesis via the pertussis toxin-sensitive G-proteins Gi/o. Thus, the effects of elevated Ca2o + concentration and all three PAMs were abolished by overnight exposure of HEK-CaSR cells to pertussis toxin. Somewhat to our surprise, however, we find that the CaSR activates rather than inhibits CREB-133 phosphorylation in both HEK-CaSR cells and human parathyroid cells and that there is a strong bias in the response in favour of elevated Ca2o + and cinacalcet but not PAMs such as L-Phe and SMG that act via the receptor's VFT domain.
doi:10.1016/j.bone.2012.08.075
O57 Is calcium-sensing receptor a double-edged sword in cancer? N. Chattopadhyay Department of Endocrinology, CSIR-Central Drug Research Institute, Chattar Manzil, Mhatma Gandhi Marg, Lucknow 226001, India Abstract: The extracellular calcium-sensing receptor (CaSR) is a major ‘sensor’ of not only extracellular Ca2 + but also polycationic molecules in the body. In the mammalian system, the CaSR serves as a major defense mechanism against hypercalcemia by sensing extracellular Ca2 + levels in the parathyroid and kidney. While mutations of CaSR gene have not yet been associated with any malignancy, anomalous functions of
Abstracts
that OS-9 may have effects on CaSR processing and expression that could profoundly influence CaSR-related disease.
doi:10.1016/j.bone.2012.08.071
O53 Calcimimetic and calcilytic drugs E.F. Nemeth MetisMedica, Toronto, Canada Abstract: Drugs acting on the calcium receptor (CaR) can rapidly alter circulating levels of parathyroid hormone (PTH) and are therefore useful in the treatment of disorders of mineral metabolism. The type II calcimimetic cinacalcet is approved for treating secondary hyperparathyroidism (HPT) in patients receiving renal replacement therapy or in those with parathyroid carcinoma and is gaining approval for treating primary HPT in a number of EU countries. The type I calcimimetic KAI-4169 is currently in late stage clinical development for treating secondary HPT in patients with chronic kidney disease, so this agonist of the CaR might join the positive allosteric modulator cinacalcet as an effective treatment for HPT. The development of most calcilytics as therapies for osteoporosis has been discontinued and it is uncertain if the failure of calcilytics to generally increase bone mineral density is due to the pharmacodynamic or pharmacokinetic properties of these compounds. In contrast to type II calcimimetics, calcilytics do not show tissue selectivity and could neutralize anabolic effects of PTH by blocking skeletal CaRs. The amino alcohol chemotype calcilytics have therefore been repositioned for new indications: ronacaleret for stem cell transplantation and NPSP790 and NPSP795 for autosomal dominant hypocalcemia. The rationale for the latter indication derives not only from the ability of calcilytics to increase PTH secretion but additionally from their ability to inhibit renal excretion of calcium. New indications for calcimimetics and calcilytics will require the discovery of compounds that preferentially act on CaRs in tissues other than the parathyroid gland and kidney.
doi:10.1016/j.bone.2012.08.072
O54 The intimate link between calcium sensing receptor trafficking and signaling M.P. Grant, A. Stepanchick, A. Cavanaugh, G.E. Breitwieser Geisinger Clinic, Danville, PA, USA Abstract: Calcium sensing receptors (CaSR) regulate systemic calcium homeostasis at the parathyroid, kidney, intestine and bone, translating fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. CaSR, a family C G protein-coupled receptor (GPCR), operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. We recently defined a unique mechanism, agonist-driven insertional signaling (ADIS), which couples plasma membrane CaSR signaling to increased anterograde trafficking of CaSR, leading to a net increase in plasma membrane CaSR during agonist exposure. Signaling is also coupled to enhanced CaSR biosynthesis, allowing maintenance of intracellular reservoirs of mobilizable receptor. We used multi-wavelength total internal reflection fluorescence (TIRF) microscopy alternating with wild-field imaging to monitor intracellular calcium. Plasma membrane CaSR undergoes constitutive endocytosis without significant recycling, thus signaling is determined by the rate of insertion of CaSR at the plasma membrane. Sustained CaSR signaling results from agonist-driven, brefeldin A-sensitive trafficking of CaSR through the secretory pathway. The intracellular reservoir of mobilizable CaSR is depleted when glycosylation of newly synthesized receptors is blocked by tunicamycin, demonstrating the coupling of receptor biosynthesis to signaling. The continuous, signaling-dependent insertion of CaSR at the plasma membrane ensures a rapid response to alterations in extracellular calcium (or allosteric agonist) concentrations despite ongoing desensitization and endocytosis. Signaling-regulated CaSR plasma membrane abundance represents a unique adaptation of the normal mechanisms regulating GPCR signaling. Supported by Geisinger Clinic and NIH.
doi:10.1016/j.bone.2012.08.073
O55 Regulation of signalling pathways by extracellular calcium in colon cancer cells S. Tennakoona, B. Marianb, J. Schmidc, E. Kállaya a Department of Pathophysiology and Allergy Research,
Medical University of Vienna, Austria Department of Internal Medicine I, Institute of Cancer Research, Medical University of Vienna, Austria c Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria b
Abstract: The calcium sensing receptor (CaSR) is a G-protein coupled receptor, which is activated by a number of ligands stimulating various downstream cell signalling pathways. In addition to its main role in regulation of calcium homeostasis, CaSR is also involved in numerous other biological processes such as cell proliferation, differentiation and lipid membrane metabolism. Previous studies demonstrated that calcium regulates cell proliferation in colonocytes but the pathways or factors involved in this process are still need to be elucidated. The aim of this study is to investigate the pathways regulated by the activated CaSR in colonocytes. In HEK cells it seems that calcium activates the MAP/ERK (mitogen-activated protein kinase/extracellular-signal-regulated kinases) and the NFkappaB (Nuclear FactorKappaB) pathways through the CaSR. Therefore we studied the impact of calcium (2 mM) on these two proliferative pathways using luciferase reporter assay and western blot in the human colon cancer cell line Caco-2. Luciferase data showed that neither the MAP/ ERK nor the NFkappaB pathway is activated by treatment with calcium in colonocytes. Further, we have seen no changes in phospho-ERK expression after treatment with calcium for 10–40 min. However we observed a reduction of phospho-ERK expression after 8 and 24 h treatment with calcium. In conclusion, our data shows that in Caco-2 cells calcium does not activate either the MAP/ERK or NFkappaB pathway and that long-term treatment may even lead to their inhibition. The next step would be to look at other candidate signalling pathways and to confirm the involvement of CaSR by modulating its expression.
doi:10.1016/j.bone.2012.08.074
O56 Modulation of cAMP and cAMP-sensitive signaling by the calcium-sensing receptor A.D. Conigravea, V.A. Avlania, A. Christopoulosb, H.-C. Muna a School of Molecular Bioscience, University of Sydney, NSW 2006, Australia b Monash Institute of Pharmaceutical Sciences, Parkville 3052 Victoria, Australia Abstract: The calcium-sensing receptor (CaSR) modulates cAMP metabolism in parathyroid cells and various other cell-types as part of the mechanism by which it provides acute control of hormone secretion. In addition, the CaSR modulates the number and differentiation state of cells in diverse tissues including osteoblasts in bone, keratinocytes in skin and intestinal epithelial cells via the activation and/or inhibition of signaling pathways upstream of key transcriptional regulators e.g., ERK1/ 2 and SRE. In recent studies, we have investigated the mechanisms by which extracellular Ca2 + and positive allosteric modulators (PAMs) of the CaSR including cinacalcet, L-Phe, and S-methylglutathione control intracellular cAMP levels as well as the impact of CaSR modulators on cAMP-dependent transcription via the transcription factor CREB. With respect to cAMP levels in CaSR-expressing HEK-293 cells and parathyroid cells, the CaSR acts primarily to suppress cAMP synthesis via the pertussis toxin-sensitive G-proteins Gi/o. Thus, the effects of elevated Ca2o + concentration and all three PAMs were abolished by overnight exposure of HEK-CaSR cells to pertussis toxin. Somewhat to our surprise, however, we find that the CaSR activates rather than inhibits CREB-133 phosphorylation in both HEK-CaSR cells and human parathyroid cells and that there is a strong bias in the response in favour of elevated Ca2o + and cinacalcet but not PAMs such as L-Phe and SMG that act via the receptor's VFT domain.
doi:10.1016/j.bone.2012.08.075
O57 Is calcium-sensing receptor a double-edged sword in cancer? N. Chattopadhyay Department of Endocrinology, CSIR-Central Drug Research Institute, Chattar Manzil, Mhatma Gandhi Marg, Lucknow 226001, India Abstract: The extracellular calcium-sensing receptor (CaSR) is a major ‘sensor’ of not only extracellular Ca2 + but also polycationic molecules in the body. In the mammalian system, the CaSR serves as a major defense mechanism against hypercalcemia by sensing extracellular Ca2 + levels in the parathyroid and kidney. While mutations of CaSR gene have not yet been associated with any malignancy, anomalous functions of