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A comparison of receptor binding, receptor activation and beta-arrestin recruitment by salmon and human calcitonin
Abstracts a
Department of Cell Biology and Anatomy, University of Turku, Finland Department of Chemistry, University of Turku, Finland c Turku PET Center, Turku University Hospital, Finland d Turku Center for Disease Modeling, University of Turku, Turku, Finland b
Abstract: MicroRNAs (miRNAs) are endogenous, small non-coding RNA molecules that bind to the 3′ untranslated region of mRNAs and induce translational repression or mRNA degradation. Chemically synthesized miRNAs have been shown to regulate gene expression and cell physiology in vitro. We studied the whole-body distribution and kinetics of intravenously administered 68Ga-labeled anti-miR15b molecules in adult male rats. Four 22-mer RNA molecules containing either seven galactoses and NOTA chelator (68Ga-NOTA-RNA-Gal), an unmodified (68Ga-NOTA-RNA), modified NOTA chelator (68Ga-NOTA(mod)-RNA) or DOTA chelator (68Ga-DOTA-RNA) were used in this study. For comparison, 68Ga-labeled NOTA-chelated oligonucleotide with six thymine residues (68Ga-NOTA-T6), the chelator (68Ga-NOTA), and the 68Galabeling reaction mixture alone were used. Each rat was given 10–20 MBq of 68Galabeled RNA, NOTA or 68Ga. Biodistribution was evaluated by performing a 60-min dynamic PET imaging with HRRT-camera (High Resolution Research Tomography). Two rats were imaged simultaneously. After PET imaging, the rats were euthanized, tissue samples were excised, weighed and measured for radioactivity using a gamma counter. 68 Ga-NOTA-RNA-Gal induced 14-fold radioactivity in liver as compared to 68GaNOTA-RNA. Both 68Ga-NOTA-RNA and 68Ga-NOTA(mod)-RNA induced multifold radioactivity in kidneys (20–30 fold), bone (4–13 fold) and bone marrow (7–23 fold), as compared with 68Ga-NOTA-T6 and 68Ga-NOTA. 68Ga-DOTA-RNA was almost identical to 68 Ga-NOTA-RNA. The 68Ga-NOTA-T6 excreted into urine, and also accumulated in small intestine and white adipose tissue. The 68Ga-NOTA accumulated in kidneys and also excreted into urine. The radioactivity derived from 68Ga reaction mixture alone had a strong tendency to accumulate in liver, spleen, lungs and bones, but not in kidneys or excrete into urine. In conclusion, the tendency of 68Ga-NOTA-RNA to accumulate in kidneys, bone and bone marrow cannot be explained by the properties of NOTA chelator. To increase uptake of 68Ga-NOTA-RNA into bone, bisphosphonates are introduced to the molecules. Further studies with bisphosphonate conjugated RNA molecules are in progress. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.269
PP081 A comparison of receptor binding, receptor activation and beta-arrestin recruitment by salmon and human calcitonin K.V. Andreassena,⁎, S.T. Petersena, M.G. Sørensenb, M. Karsdalc, K. Henriksenb a Diabetes Group, Nordic Bioscience, Herlev, Denmark b Bone Group, Nordic Bioscience, Herlev, Denmark c R&D, Nordic Bioscience, Herlev, Denmark Abstract: The calcitonins are a group of evolutionary conserved 32 amino acid peptide hormones. They have been divided into distinct groups of which the human/ rodent and the teleost/avian have been studied the most. It is well established that the human/rat and teleost/avian groups are different at least three levels related to the calcitonin receptor (CTR). These are ligand potency, ligand/receptor dissociation and ligand-induced CTR conformational changes. Hence, it is quite possible that these differences will have an effect in the signal transduction of the CTR. As there are indications of differences in terms of ligand potency and binding time, we set out to investigate whether sCT provides a different response than human calcitonin (hCT) in terms of the quality of the downstream signals mediated by the two ligands by measuring cAMP induction, beta-arrestin recruitment and ligand dissociation. This was investigated in human CTRa overexpressing COS-7 1375.10 cells and U2OS CALCR cells. The cAMP induction was measured with cAMP femto Tb kit and beta-arrestin recruitment was measured using enzyme complementation. Ligand binding was investigated by measuring the release of I-125 labeled sCT and hCT to the culture medium after 1 h incubation with the I-125 labeled ligands. All experiments were conducted on live cells by profiling sCT and hCT dose responses for short time stimulation (1 h or less) as well as for long term stimulation (up to 72 h). Our results indicate that sCT has a distinct profile from hCT during prolonged exposure to the ligand, whereas short term exposure resulted in almost identical profiles. We have shown that sCT induces a stronger and more prolonged response on both cAMP (50% increase after 8 h) and beta-arrestin levels (10 fold increase after 48 h) compared to hCT. Furthermore, ligand release experiments show a half-life of 8 h for sCT ligand release into the supernatant compared to a half-life of 3 h for hCT. In addition, inhibiting the proteolytic activity of lysosomes with 200 nM bafilomycin
S91
altered the hCT beta-arrestin recruitment response as well as the ligand release towards a sCT-like response. All this suggests a different mode of action by sCT, possibly by changing the intracellular trafficking of internalized CTRs towards increased recycling, whereas hCT drives the internalization towards degradation. These indications could have implications for the use of calcitonins in therapy of osteoporosis. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: K. V. Andreassen: none declared, S. T. Petersen: none declared, M. G. Sørensen: none declared, M. Karsdal: shareholder of Nordic Bioscience, K. Henriksen: none declared.
doi:10.1016/j.bone.2012.02.270
PP082 Visualisation of the apical surface of activated osteoclasts K. Szewczyk⁎, K. Fuller, R. Moss, T. Chambers St George's University, London, UK
Abstract: Bone resorption occurs at the substrate-apposed, apical surface of osteoclasts, a surface that has not been accessible to direct visualisation. We have recently shown that it is the high affinity of bone mineral for vitronectin-receptor ligands that endows bone with the ability to activate osteoclasts. Consistent with this, osteoclasts secrete acid hydrolases, relocate proton pumps to the apical surface, and develop a ruffled border, as they do on bone, when they are incubated on vitronectincoated plastic. Therefore, osteoclasts can be activated for resorption by substrates other than bone, so long as the surface is coated with ligands for the vitronectin receptor. To further analyse the apical surface of activated osteoclasts, we exploited an approach that enables inspection of the substrate-apposed surface of cells. For this, glass slides are coated with nail varnish and then adhesive ligands. After incubation of cells on this surface, the discs of nail varnish are inverted onto a glass slide. The nail varnish is then removed with acetone, exposing the underside of the cells for inspection in the scanning electron microscope. Vitronectin induced a striking appearance in the underside of osteoclasts. The circumferential region was flat and featureless unless osmium tetroxide was omitted from fixation. Omission of osmium tetroxide revealed circumferential rings and crescents of nodular protrusions, corresponding in position with podosomes. The surface between and adjacent to podosomes, overlying the peri-podosomal cytoskeletal network, was extensively pitted. Within the circumferential region, zones of compacted, fine, finger-like processes were seen, most commonly as islands, or rings at the cell periphery, but sometimes throughout the whole central region. In the confocal microscope, these zones corresponded in distribution to zones in which proton pumps were present at the cell apex. Some areas of the apical surface, especially towards the retreating pole of the cell, showed zones of less tightly packed processes with sucker-like apices. Elsewhere, the apical surface showed orifices, which presumably represent areas of endocytosis or exocytosis. This approach provides an exciting opportunity to characterise the morphological correlates of the resorptive process in osteoclasts. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.271
PP083 The gut microbiota regulates bone mass in mice K. Sjogrena,⁎, C. Engdahla, P. Henninga, U.H. Lernera, V. Tremarolib, M.K. Lagerquista, F. Bäckhedb, C. Ohlssona a Centre for Bone and Mineral Research, Medicine, Göteborg, Sweden b Cardiovascular and Metabolic Research, Medicine, Göteborg, Sweden Abstract: The gut microbiota modulates host metabolism and development of immune status. Here we show that the gut microbiota is also a major regulator of bone mass in mice. Germ-free (GF) mice exhibit increased bone mass associated with reduced number of osteoclasts per bone surface compared with conventionally raised (CONV-R) mice. Colonization of GF mice with a normal gut microbiota at 3 weeks of age normalizes bone mass. Furthermore, GF mice have decreased frequency of CD4+ T-cells and CD11b+/GR 1− osteoclast precursor cells in bone marrow which could also be normalized by colonization. GF mice exhibited reduced expression of the inflammatory cytokines IL-6 and TNFα in bone compared to CONV-R mice. In summary, the gut microbiota regulates bone mass in mice and we provide evidence for a mechanism involving altered immune status in bone and thereby affected osteoclast-
Department of Cell Biology and Anatomy, University of Turku, Finland Department of Chemistry, University of Turku, Finland c Turku PET Center, Turku University Hospital, Finland d Turku Center for Disease Modeling, University of Turku, Turku, Finland b
Abstract: MicroRNAs (miRNAs) are endogenous, small non-coding RNA molecules that bind to the 3′ untranslated region of mRNAs and induce translational repression or mRNA degradation. Chemically synthesized miRNAs have been shown to regulate gene expression and cell physiology in vitro. We studied the whole-body distribution and kinetics of intravenously administered 68Ga-labeled anti-miR15b molecules in adult male rats. Four 22-mer RNA molecules containing either seven galactoses and NOTA chelator (68Ga-NOTA-RNA-Gal), an unmodified (68Ga-NOTA-RNA), modified NOTA chelator (68Ga-NOTA(mod)-RNA) or DOTA chelator (68Ga-DOTA-RNA) were used in this study. For comparison, 68Ga-labeled NOTA-chelated oligonucleotide with six thymine residues (68Ga-NOTA-T6), the chelator (68Ga-NOTA), and the 68Galabeling reaction mixture alone were used. Each rat was given 10–20 MBq of 68Galabeled RNA, NOTA or 68Ga. Biodistribution was evaluated by performing a 60-min dynamic PET imaging with HRRT-camera (High Resolution Research Tomography). Two rats were imaged simultaneously. After PET imaging, the rats were euthanized, tissue samples were excised, weighed and measured for radioactivity using a gamma counter. 68 Ga-NOTA-RNA-Gal induced 14-fold radioactivity in liver as compared to 68GaNOTA-RNA. Both 68Ga-NOTA-RNA and 68Ga-NOTA(mod)-RNA induced multifold radioactivity in kidneys (20–30 fold), bone (4–13 fold) and bone marrow (7–23 fold), as compared with 68Ga-NOTA-T6 and 68Ga-NOTA. 68Ga-DOTA-RNA was almost identical to 68 Ga-NOTA-RNA. The 68Ga-NOTA-T6 excreted into urine, and also accumulated in small intestine and white adipose tissue. The 68Ga-NOTA accumulated in kidneys and also excreted into urine. The radioactivity derived from 68Ga reaction mixture alone had a strong tendency to accumulate in liver, spleen, lungs and bones, but not in kidneys or excrete into urine. In conclusion, the tendency of 68Ga-NOTA-RNA to accumulate in kidneys, bone and bone marrow cannot be explained by the properties of NOTA chelator. To increase uptake of 68Ga-NOTA-RNA into bone, bisphosphonates are introduced to the molecules. Further studies with bisphosphonate conjugated RNA molecules are in progress. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.269
PP081 A comparison of receptor binding, receptor activation and beta-arrestin recruitment by salmon and human calcitonin K.V. Andreassena,⁎, S.T. Petersena, M.G. Sørensenb, M. Karsdalc, K. Henriksenb a Diabetes Group, Nordic Bioscience, Herlev, Denmark b Bone Group, Nordic Bioscience, Herlev, Denmark c R&D, Nordic Bioscience, Herlev, Denmark Abstract: The calcitonins are a group of evolutionary conserved 32 amino acid peptide hormones. They have been divided into distinct groups of which the human/ rodent and the teleost/avian have been studied the most. It is well established that the human/rat and teleost/avian groups are different at least three levels related to the calcitonin receptor (CTR). These are ligand potency, ligand/receptor dissociation and ligand-induced CTR conformational changes. Hence, it is quite possible that these differences will have an effect in the signal transduction of the CTR. As there are indications of differences in terms of ligand potency and binding time, we set out to investigate whether sCT provides a different response than human calcitonin (hCT) in terms of the quality of the downstream signals mediated by the two ligands by measuring cAMP induction, beta-arrestin recruitment and ligand dissociation. This was investigated in human CTRa overexpressing COS-7 1375.10 cells and U2OS CALCR cells. The cAMP induction was measured with cAMP femto Tb kit and beta-arrestin recruitment was measured using enzyme complementation. Ligand binding was investigated by measuring the release of I-125 labeled sCT and hCT to the culture medium after 1 h incubation with the I-125 labeled ligands. All experiments were conducted on live cells by profiling sCT and hCT dose responses for short time stimulation (1 h or less) as well as for long term stimulation (up to 72 h). Our results indicate that sCT has a distinct profile from hCT during prolonged exposure to the ligand, whereas short term exposure resulted in almost identical profiles. We have shown that sCT induces a stronger and more prolonged response on both cAMP (50% increase after 8 h) and beta-arrestin levels (10 fold increase after 48 h) compared to hCT. Furthermore, ligand release experiments show a half-life of 8 h for sCT ligand release into the supernatant compared to a half-life of 3 h for hCT. In addition, inhibiting the proteolytic activity of lysosomes with 200 nM bafilomycin
S91
altered the hCT beta-arrestin recruitment response as well as the ligand release towards a sCT-like response. All this suggests a different mode of action by sCT, possibly by changing the intracellular trafficking of internalized CTRs towards increased recycling, whereas hCT drives the internalization towards degradation. These indications could have implications for the use of calcitonins in therapy of osteoporosis. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: K. V. Andreassen: none declared, S. T. Petersen: none declared, M. G. Sørensen: none declared, M. Karsdal: shareholder of Nordic Bioscience, K. Henriksen: none declared.
doi:10.1016/j.bone.2012.02.270
PP082 Visualisation of the apical surface of activated osteoclasts K. Szewczyk⁎, K. Fuller, R. Moss, T. Chambers St George's University, London, UK
Abstract: Bone resorption occurs at the substrate-apposed, apical surface of osteoclasts, a surface that has not been accessible to direct visualisation. We have recently shown that it is the high affinity of bone mineral for vitronectin-receptor ligands that endows bone with the ability to activate osteoclasts. Consistent with this, osteoclasts secrete acid hydrolases, relocate proton pumps to the apical surface, and develop a ruffled border, as they do on bone, when they are incubated on vitronectincoated plastic. Therefore, osteoclasts can be activated for resorption by substrates other than bone, so long as the surface is coated with ligands for the vitronectin receptor. To further analyse the apical surface of activated osteoclasts, we exploited an approach that enables inspection of the substrate-apposed surface of cells. For this, glass slides are coated with nail varnish and then adhesive ligands. After incubation of cells on this surface, the discs of nail varnish are inverted onto a glass slide. The nail varnish is then removed with acetone, exposing the underside of the cells for inspection in the scanning electron microscope. Vitronectin induced a striking appearance in the underside of osteoclasts. The circumferential region was flat and featureless unless osmium tetroxide was omitted from fixation. Omission of osmium tetroxide revealed circumferential rings and crescents of nodular protrusions, corresponding in position with podosomes. The surface between and adjacent to podosomes, overlying the peri-podosomal cytoskeletal network, was extensively pitted. Within the circumferential region, zones of compacted, fine, finger-like processes were seen, most commonly as islands, or rings at the cell periphery, but sometimes throughout the whole central region. In the confocal microscope, these zones corresponded in distribution to zones in which proton pumps were present at the cell apex. Some areas of the apical surface, especially towards the retreating pole of the cell, showed zones of less tightly packed processes with sucker-like apices. Elsewhere, the apical surface showed orifices, which presumably represent areas of endocytosis or exocytosis. This approach provides an exciting opportunity to characterise the morphological correlates of the resorptive process in osteoclasts. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.271
PP083 The gut microbiota regulates bone mass in mice K. Sjogrena,⁎, C. Engdahla, P. Henninga, U.H. Lernera, V. Tremarolib, M.K. Lagerquista, F. Bäckhedb, C. Ohlssona a Centre for Bone and Mineral Research, Medicine, Göteborg, Sweden b Cardiovascular and Metabolic Research, Medicine, Göteborg, Sweden Abstract: The gut microbiota modulates host metabolism and development of immune status. Here we show that the gut microbiota is also a major regulator of bone mass in mice. Germ-free (GF) mice exhibit increased bone mass associated with reduced number of osteoclasts per bone surface compared with conventionally raised (CONV-R) mice. Colonization of GF mice with a normal gut microbiota at 3 weeks of age normalizes bone mass. Furthermore, GF mice have decreased frequency of CD4+ T-cells and CD11b+/GR 1− osteoclast precursor cells in bone marrow which could also be normalized by colonization. GF mice exhibited reduced expression of the inflammatory cytokines IL-6 and TNFα in bone compared to CONV-R mice. In summary, the gut microbiota regulates bone mass in mice and we provide evidence for a mechanism involving altered immune status in bone and thereby affected osteoclast-