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Osteoclast proton transport modulation by fluid flow
Abstracts / Bone 48 (2011) S124–S137 c
School of Medical Sciences, Sydney, Australia Faculty of Medicine, University of New South Wales, Sydney, Australia e Clinical School, St Vincent's Hospital, Sydney, Australia d
Abstract: Neuropeptide Y (NPY) has been demonstrated to regulate osteoblast activity through a signalling pathway involving hypothalamic and local osteoblastic signalling. In contrast, the potential action of NPY on bone resorption has not been elucidated. Recent data however, indicate that NPY may also regulate the osteoclast, through specific NPY receptors, most likely signalling via Y1 and Y6 receptors. NPY, Y6 receptors are mainly expressed in the hypothalamus, and are the least studied Y receptor. In Y6 KO mice femoral BMD (47.1±1.1 mg/cm2 vs 57.9±2.4, p<0.01) and BMC (18.6± 1.0 mg vs 23.7±1.0, p<0.05) were reduced compared to wild type. Cancellous bone volume was also significantly decreased in Y6 KO compared to wild type (7.3±0.5% vs 10.1±0.7, p<0.01), with fewer trabeculae (1.4±0.1 mm− 1 vs 1.8±0.1, p<0.01) without a difference in trabecular thickness (53.5±1 mm vs 55.6±1). Importantly, this effect was associated with greater osteoclast surface (19.7±1.7% vs 12.5±1.3, p<0.02) and osteoclast number (8.5±0.6 mm− 1 vs 4.2±0.6, p<0.002), without any change in bone formation (data not shown). These data indicate that NPY regulates osteoclastic cells via a centrally mediated pathway. In addition, NPY, Y1 receptors regulate bone formation by direct action in osteoblasts, however, germline deletion of the Y1 receptor results in greater osteoclast surface. In contrast, deletion of osteoblastic Y1 receptors or those from the hypothalamus does not alter osteoclast surface and the RANKL/OPG ratio is unaltered in both germline and osteoblastic Y1 KO mice, indicating a non-osteoblastic, potentially direct osteoclastic process. Exploring this possibility in a mouse model with deletion of Y1 in cells of the macrophage lineage, including osteoclasts, there was a trend toward decreased bone mass, with a 10% reduction in femoral BMC (25.5 ±0.1 mg vs 28.5 ±1.8, p= 0.18). Taken together, these findings from Y6 KO and Y1 KO models indicate that bone resorption is regulated by neural pathways mediated by NPY signalling. The purely hypothalamic expression of Y6 receptors indicates a centrally mediated pathway to the osteoclast. In contrast, the Y1 receptor models suggest a peripheral, non-osteoblastic and potentially direct effect on the osteoclast. Thus, as has been shown for bone formation, bone resorption is regulated by NPY signalling via both central and peripheral receptors. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: N. Lee: None Declared, F. Driessler: None Declared, A. Zengin: None Declared, I. Wong: None Declared, E. Yulyaningsih : None Declared, R. Enriquez : None Declared, A. Sainsbury : None Declared, H. Herzog: None Declared, J. Eisman Grant / Research Support from Amgen, Eli Lilly, Merck Sharp & Dohme, Novartis, Sanofi-Aventis, Servier, Advisory Board Membership of Amgen, Eli Lilly, Merck Sharp & Dohme, Novartis, SanofiAventis, Servier, Consulting fees from Amgen, deCode, Eli Lilly, Merck Sharp & Dohme, Novartis, Sanofi-Aventis, Servier, P. Baldock : None Declared. doi:10.1016/j.bone.2011.03.275
PP117-T Stimulation of mouse calvarial periosteal resorption and inhibition of osteoclast formation in human peripheral CD14+ cells by vitamin A P. Henning a,⁎, A. Pirhayati b, U. Pettersson c, O. Svensson d, H.H. Conaway e, U. Lerner a,b a Centre for Bone and Arthitis Research at Institute for Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Umeå, Sweden b Oral Cell Biology, Umeå University, Umeå, Sweden c Clinical Pharmacology, Umeå University, Umeå, Sweden d Orthopedics, Umeå University, Umeå, Sweden e Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, USA Abstract: Increased vitamin A intake has been associated with enhanced cortical bone resorption, decreased bone mass, and increased fractures. We have recently reported that vitamin A is a potent inhibitor of RANKL induced osteoclast formation in mouse bone marrow macrophages (Faseb J 23:3526, 2009). In the present study, we have investigated effects of retinoids on bone resorption in mouse calvarial bones and osteoclast differentiation in human CD14+ peripheral blood monocytes. All-trans-retinoic acid (ATRA) enhanced 45Ca release in a time and concentration dependent manner in mouse calvariae. Retinol, retinalaldehyde, and 9-cis-retinoid acid also stimulated 45Ca release. The resorptive effect of ATRA was characterized by enhanced bone matrix degradation, increased mRNA expression of genes associated with osteoclast differentiation (calcitonin receptor, cathepsin K and TRAP), and inhibition by calcitonin and bisphosphonate. ATRA stimulated RANKL mRNA and protein and decreased OPG mRNA and protein at concentrations similar to those stimulating 45 Ca release. The 45Ca release induced by ATRA was blocked by OPG. Retinoid acid receptors (RARa/b/g) were expressed in the calvarial bones. RARa agonists enhanced the release of 45 Ca, increased the mRNA expression of rank, and decreased the mRNA expression of opg. RARb or RARb/g agonists had no effects. In contrast to the stimulatory effects in calvariae, ATRA inhibited RANKL induced osteoclast formation in human CD14+ cell cultures. The inhibition was associated with decreased mRNA expression of cathepsin K and trap and
S135
mimicked by panRAR agonists and a specific RARα agonist, but not by agonists for RARβ or RARβ/γ. Inhibition of osteoclast formation by ATRA was reversed by a RARα antagonist. Based on their capacity to phagocytose zymosan, RANKL stimulated CD14+ cells treated with ATRA exhibited a macrophage phenotype. These data show two contrasting actions of ATRA mediated by RARα receptors: 1. stimulation of neonatal mouse periosteal bone resorption, and 2. inhibition of RANKL induced osteoclast differentiation of CD14+ peripheral monocytes. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.276
PP118-S Osteoclast proton transport modulation by fluid flow P. Morethson⁎ Physiology and Biophysics, University of São Paulo, São Paulo, Brazil Abstract: During bone resorption, proton secretion by the osteoclast is a necessary step for bone mineral dissolution. Considering that our previous work regarding osteoclast proton secretion and intracellular pH (pHi) regulation showed that the pHi of these cells is not stable, but seems to oscillate in time, exhibiting periods of spontaneous intracellular alkalinization and acidification [Morethson P, Musa-Aziz R, 2010. Spontaneous rhythmic fluctuations of osteoclasts intracellular pH. J Bone Miner Res 25 (Suppl 1)], this work was performed to evaluate the effect of the fluid flow presence or absence in the osteoclast H+ transport mechanisms possibly related to spontaneous pHi variations. To address this question, we worked with freshly isolated osteoclasts from long bones or osteoclast-like (OCL) cells generated from bone marrow precursor cells of Wistar rats (using M-CSF 50 ng/ml and RANK-L 10 ng/ml). Cells were maintained onto plastic coverslips in α-MEM+ 10% FBS at pH 7.4, placed in a 5% CO2 incubator at 37 °C. The pHi measurements were performed by fluorescence microscopy using BCECF-AM (12 mM), a fluorescent probe sensitive to the pHi. The emitted fluorescence (EF) ratios (R) after excitation at 490 and 440 nm (R = EF490/EFI440) were converted to pHi using the high-K+ nigericin technique. The experiments were performed in the presence of standard HEPES solution nominally free of CO2/HCO− 3 at pH 7.4, 300 mOsm/L H2O, at 37 °C. During fluorescence records, we tested the effect of the cessation of the fluid flow after a period of continuous perfusion and vice-versa. Our results suggest that, in our experimental conditions, the cessation of the fluid flow initiates progressive intracellular alkalinization (n= 8). Inversely, after a period of interrupted flow, the initiation of a continuous perfusion leads the osteoclasts and OCL-cells pHi to progressive acidification (n = 6). These data may suggest possible modulation of the bone resorption by the fluid flow at the interface osteoclast/bone interstitial fluid. This modulation could be relevant in pathological conditions in which there are changes in blood pressure or bone vascularization. (Supported by: CNPq, Fapesp). This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.277
PP119-M Semi-automated histomorphometry of bone resorption parameters R.J. Van 't Hof ⁎, E. Landao-Basonga Rheumatic Diseases Unit, University of Edinburgh, Edinburgh, UK Abstract: Micro-CT analysis has become the gold standard for assessing bone volume and architecture in small animals. However, micro-CT does not allow the assessment of bone turnover parameters such as bone formation rate and osteoclast (OC) numbers and surfaces. For these crucial parameters histomorphometric analysis is still an essential technique. Histomorphometry however, is a time consuming technique and, especially in the mouse, OC can be hard to detect. Here we present a rapid semi-automated analysis of histomorphometric bone resorption parameters. Mouse femurs and vertebrae were fixed for 24 h in neutral buffered formalin and embedded in methyl methacrylate. Sections (5 μm) were prepared, OCs identified by TRAcP staining, and bone stained using aniline blue. Sections were imaged on a Zeis Axioimager microscope fitted with a QImaging Retiga 4000 camera, calibrated using a stage micrometer. Multiple images of a section were combined using the 2D stitching plugin in ImageJ. Analysis software was written in Java using the Netbeans IDE and ImageJ as an image analysis library. Both the source code and the final program are available free from the authors. The combination of the aniline blue and TRAcP stains results in blue-stained bone tissue and red-stained OCs, a result which is relatively easily analysed using simple thresholding. The final analysis program starts with identifying the bone tissue by an interactive threshold in the huesaturation-brightness (HSB) colour space. Small sectioning artifacts such as cracks can be removed using simple binary close and holefill operations. Small particles in the image are removed using an erode-reconstruct algorithm. Next, the OCs are identified using a threshold operation. As OCs are normally located on the bone surface, the user can specify a maximum distance from the bone surface. TRAcP positive fragments that are in close proximity are likely to be part of a single OC. A binary closing operation can merge these fragments into single objects. The final results are saved in an Excel compatible file. The attached image shows a section with OCs identified in red, OC surface in magenta and bone surface in blue.
School of Medical Sciences, Sydney, Australia Faculty of Medicine, University of New South Wales, Sydney, Australia e Clinical School, St Vincent's Hospital, Sydney, Australia d
Abstract: Neuropeptide Y (NPY) has been demonstrated to regulate osteoblast activity through a signalling pathway involving hypothalamic and local osteoblastic signalling. In contrast, the potential action of NPY on bone resorption has not been elucidated. Recent data however, indicate that NPY may also regulate the osteoclast, through specific NPY receptors, most likely signalling via Y1 and Y6 receptors. NPY, Y6 receptors are mainly expressed in the hypothalamus, and are the least studied Y receptor. In Y6 KO mice femoral BMD (47.1±1.1 mg/cm2 vs 57.9±2.4, p<0.01) and BMC (18.6± 1.0 mg vs 23.7±1.0, p<0.05) were reduced compared to wild type. Cancellous bone volume was also significantly decreased in Y6 KO compared to wild type (7.3±0.5% vs 10.1±0.7, p<0.01), with fewer trabeculae (1.4±0.1 mm− 1 vs 1.8±0.1, p<0.01) without a difference in trabecular thickness (53.5±1 mm vs 55.6±1). Importantly, this effect was associated with greater osteoclast surface (19.7±1.7% vs 12.5±1.3, p<0.02) and osteoclast number (8.5±0.6 mm− 1 vs 4.2±0.6, p<0.002), without any change in bone formation (data not shown). These data indicate that NPY regulates osteoclastic cells via a centrally mediated pathway. In addition, NPY, Y1 receptors regulate bone formation by direct action in osteoblasts, however, germline deletion of the Y1 receptor results in greater osteoclast surface. In contrast, deletion of osteoblastic Y1 receptors or those from the hypothalamus does not alter osteoclast surface and the RANKL/OPG ratio is unaltered in both germline and osteoblastic Y1 KO mice, indicating a non-osteoblastic, potentially direct osteoclastic process. Exploring this possibility in a mouse model with deletion of Y1 in cells of the macrophage lineage, including osteoclasts, there was a trend toward decreased bone mass, with a 10% reduction in femoral BMC (25.5 ±0.1 mg vs 28.5 ±1.8, p= 0.18). Taken together, these findings from Y6 KO and Y1 KO models indicate that bone resorption is regulated by neural pathways mediated by NPY signalling. The purely hypothalamic expression of Y6 receptors indicates a centrally mediated pathway to the osteoclast. In contrast, the Y1 receptor models suggest a peripheral, non-osteoblastic and potentially direct effect on the osteoclast. Thus, as has been shown for bone formation, bone resorption is regulated by NPY signalling via both central and peripheral receptors. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: N. Lee: None Declared, F. Driessler: None Declared, A. Zengin: None Declared, I. Wong: None Declared, E. Yulyaningsih : None Declared, R. Enriquez : None Declared, A. Sainsbury : None Declared, H. Herzog: None Declared, J. Eisman Grant / Research Support from Amgen, Eli Lilly, Merck Sharp & Dohme, Novartis, Sanofi-Aventis, Servier, Advisory Board Membership of Amgen, Eli Lilly, Merck Sharp & Dohme, Novartis, SanofiAventis, Servier, Consulting fees from Amgen, deCode, Eli Lilly, Merck Sharp & Dohme, Novartis, Sanofi-Aventis, Servier, P. Baldock : None Declared. doi:10.1016/j.bone.2011.03.275
PP117-T Stimulation of mouse calvarial periosteal resorption and inhibition of osteoclast formation in human peripheral CD14+ cells by vitamin A P. Henning a,⁎, A. Pirhayati b, U. Pettersson c, O. Svensson d, H.H. Conaway e, U. Lerner a,b a Centre for Bone and Arthitis Research at Institute for Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Umeå, Sweden b Oral Cell Biology, Umeå University, Umeå, Sweden c Clinical Pharmacology, Umeå University, Umeå, Sweden d Orthopedics, Umeå University, Umeå, Sweden e Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, USA Abstract: Increased vitamin A intake has been associated with enhanced cortical bone resorption, decreased bone mass, and increased fractures. We have recently reported that vitamin A is a potent inhibitor of RANKL induced osteoclast formation in mouse bone marrow macrophages (Faseb J 23:3526, 2009). In the present study, we have investigated effects of retinoids on bone resorption in mouse calvarial bones and osteoclast differentiation in human CD14+ peripheral blood monocytes. All-trans-retinoic acid (ATRA) enhanced 45Ca release in a time and concentration dependent manner in mouse calvariae. Retinol, retinalaldehyde, and 9-cis-retinoid acid also stimulated 45Ca release. The resorptive effect of ATRA was characterized by enhanced bone matrix degradation, increased mRNA expression of genes associated with osteoclast differentiation (calcitonin receptor, cathepsin K and TRAP), and inhibition by calcitonin and bisphosphonate. ATRA stimulated RANKL mRNA and protein and decreased OPG mRNA and protein at concentrations similar to those stimulating 45 Ca release. The 45Ca release induced by ATRA was blocked by OPG. Retinoid acid receptors (RARa/b/g) were expressed in the calvarial bones. RARa agonists enhanced the release of 45 Ca, increased the mRNA expression of rank, and decreased the mRNA expression of opg. RARb or RARb/g agonists had no effects. In contrast to the stimulatory effects in calvariae, ATRA inhibited RANKL induced osteoclast formation in human CD14+ cell cultures. The inhibition was associated with decreased mRNA expression of cathepsin K and trap and
S135
mimicked by panRAR agonists and a specific RARα agonist, but not by agonists for RARβ or RARβ/γ. Inhibition of osteoclast formation by ATRA was reversed by a RARα antagonist. Based on their capacity to phagocytose zymosan, RANKL stimulated CD14+ cells treated with ATRA exhibited a macrophage phenotype. These data show two contrasting actions of ATRA mediated by RARα receptors: 1. stimulation of neonatal mouse periosteal bone resorption, and 2. inhibition of RANKL induced osteoclast differentiation of CD14+ peripheral monocytes. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.276
PP118-S Osteoclast proton transport modulation by fluid flow P. Morethson⁎ Physiology and Biophysics, University of São Paulo, São Paulo, Brazil Abstract: During bone resorption, proton secretion by the osteoclast is a necessary step for bone mineral dissolution. Considering that our previous work regarding osteoclast proton secretion and intracellular pH (pHi) regulation showed that the pHi of these cells is not stable, but seems to oscillate in time, exhibiting periods of spontaneous intracellular alkalinization and acidification [Morethson P, Musa-Aziz R, 2010. Spontaneous rhythmic fluctuations of osteoclasts intracellular pH. J Bone Miner Res 25 (Suppl 1)], this work was performed to evaluate the effect of the fluid flow presence or absence in the osteoclast H+ transport mechanisms possibly related to spontaneous pHi variations. To address this question, we worked with freshly isolated osteoclasts from long bones or osteoclast-like (OCL) cells generated from bone marrow precursor cells of Wistar rats (using M-CSF 50 ng/ml and RANK-L 10 ng/ml). Cells were maintained onto plastic coverslips in α-MEM+ 10% FBS at pH 7.4, placed in a 5% CO2 incubator at 37 °C. The pHi measurements were performed by fluorescence microscopy using BCECF-AM (12 mM), a fluorescent probe sensitive to the pHi. The emitted fluorescence (EF) ratios (R) after excitation at 490 and 440 nm (R = EF490/EFI440) were converted to pHi using the high-K+ nigericin technique. The experiments were performed in the presence of standard HEPES solution nominally free of CO2/HCO− 3 at pH 7.4, 300 mOsm/L H2O, at 37 °C. During fluorescence records, we tested the effect of the cessation of the fluid flow after a period of continuous perfusion and vice-versa. Our results suggest that, in our experimental conditions, the cessation of the fluid flow initiates progressive intracellular alkalinization (n= 8). Inversely, after a period of interrupted flow, the initiation of a continuous perfusion leads the osteoclasts and OCL-cells pHi to progressive acidification (n = 6). These data may suggest possible modulation of the bone resorption by the fluid flow at the interface osteoclast/bone interstitial fluid. This modulation could be relevant in pathological conditions in which there are changes in blood pressure or bone vascularization. (Supported by: CNPq, Fapesp). This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.277
PP119-M Semi-automated histomorphometry of bone resorption parameters R.J. Van 't Hof ⁎, E. Landao-Basonga Rheumatic Diseases Unit, University of Edinburgh, Edinburgh, UK Abstract: Micro-CT analysis has become the gold standard for assessing bone volume and architecture in small animals. However, micro-CT does not allow the assessment of bone turnover parameters such as bone formation rate and osteoclast (OC) numbers and surfaces. For these crucial parameters histomorphometric analysis is still an essential technique. Histomorphometry however, is a time consuming technique and, especially in the mouse, OC can be hard to detect. Here we present a rapid semi-automated analysis of histomorphometric bone resorption parameters. Mouse femurs and vertebrae were fixed for 24 h in neutral buffered formalin and embedded in methyl methacrylate. Sections (5 μm) were prepared, OCs identified by TRAcP staining, and bone stained using aniline blue. Sections were imaged on a Zeis Axioimager microscope fitted with a QImaging Retiga 4000 camera, calibrated using a stage micrometer. Multiple images of a section were combined using the 2D stitching plugin in ImageJ. Analysis software was written in Java using the Netbeans IDE and ImageJ as an image analysis library. Both the source code and the final program are available free from the authors. The combination of the aniline blue and TRAcP stains results in blue-stained bone tissue and red-stained OCs, a result which is relatively easily analysed using simple thresholding. The final analysis program starts with identifying the bone tissue by an interactive threshold in the huesaturation-brightness (HSB) colour space. Small sectioning artifacts such as cracks can be removed using simple binary close and holefill operations. Small particles in the image are removed using an erode-reconstruct algorithm. Next, the OCs are identified using a threshold operation. As OCs are normally located on the bone surface, the user can specify a maximum distance from the bone surface. TRAcP positive fragments that are in close proximity are likely to be part of a single OC. A binary closing operation can merge these fragments into single objects. The final results are saved in an Excel compatible file. The attached image shows a section with OCs identified in red, OC surface in magenta and bone surface in blue.