Sensing Receptor Research Articles

A Novel Role for the Calcium Sensing Receptor in Rat Diabetic Encephalopathy

Background: Diabetic encephalopathy is a common complication of diabetes, and it may be involved in altering intracellular calcium concentrations ([Ca²⁺]_i) at its onset. The calcium sensing receptor (CaSR) is a G-protein coupled receptor, however, the functional involvement of CaSR in diabetic encephalopathy remains unclear. Methods: In this study, diabetic rats were modeled by STZ (50 mg/kg). At the end of 4, 8 and 12 weeks, the CaSR expression in hippocampus was analyzed by Western blot. In neonatal rat hippocampal neurons, the [Ca²⁺]_i was detected by laser scanning confocal microscopy, the production of reactive oxygen species (ROS) in mitochondria, the level of NO and the mitochondrial transmembrane potential were measured by MitoSOX, DAF-FM and JC-1, respectively. Results: Our results showed in hippocampal neurons treated with high glucose, CaSR regulated [Ca²⁺]_i through the PLC-IP3 pathway. CaSR expression was decreased and was involved in the changes in [Ca²⁺]_i. Mitochondrial membrane potential, NO release and expression of p-eNOS decreased, while the production of ROS in mitochondria increased. Conclusion: Down-regulation of CaSR expression was accompanied by neuronal injury, calcium disturbance, increased ROS production and decreased release of NO. Up-regulation of CaSR expression attenuated these changes through a positive compensatory protective mechanism to inhibit and delay diabetic encephalopathy in rats.

R990G polymorphism of calcium sensing receptor gene is associated with high parathyroid hormone levels in subjects with vitamin D deficiency: a cross-sectional study.

Single nucleotide polymorphisms (SNPs), R990G and A986S of the calcium sensing receptor (CaSR) gene, are shown to influence response of parathyroid hormone (PTH) in subjects with optimal vitamin D levels. This cross-sectional study was conducted in subjects with vitamin D deficiency (VDD) to observe associations between CaSR polymorphisms, plasma iPTH, and serum calcium levels. Adult females (n = 140) with known VDD, intact parathyroid hormone (iPTH), and calcium levels were recruited for genotype analysis. The frequencies of the 986 alleles GG, GT, and TT were 68%, 25%, and 7%, respectively, whereas the frequencies of the 990 alleles AA, AG, and GG were 80%, 8.9%, and 11.1%, respectively. The subjects with GG genotype of R990G polymorphism had higher iPTH levels (148.65 versus 91.47 and 86.1 pg/mL for GG versus AA, AG, resp., P = 0.008) and lower calcium levels (8.4 versus 9.04 and 9.07 mg/dL for GG versus AA, AG, resp., P = 0.002). No such association of A986S polymorphism with plasma iPTH or serum calcium levels was observed in the present study. Patients with VDD bearing the GG genotype of R990G SNPs are prone to have higher iPTH levels and lower calcium.

Calcium sensing receptor activation by calcimimetic R-568 in human amniotic fluid mesenchymal stem cells: correlation with osteogenic differentiation.

Human amniotic fluid mesenchymal stem cells (hAFMSCs) are promising for therapeutic applications in bone damage. Calcium sensing receptor (CaSR), a G protein-coupled receptor, plays a physiological role in the regulation of bone metabolism. Thus, the bone CaSR could be targeted by calcimimetic agonists, which may be potentially helpful in treating bone diseases. The aim of our study was to characterize CaSR expression in hAFMSCs and to assess the activity of calcimimetic R-568 during in vitro osteogenesis. Using western blotting, immunofluorescence, and flow cytometry, we consistently observed constitutive CaSR in osteo-differentiating hAFMSCs. Notably, both R-568 and calcium significantly enhanced hAFMSC osteogenic differentiation after exposure to osteogenic medium. To provide further evidence of the involvement of CaSR in osteogenesis, we correlated its expression with that of established osteogenic markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN), and novel, not yet completely defined regulators of osteogenesis. Among these are β-catenin and Slug, which are mediators of Wnt signaling, and nuclear factor of activated T cells c1 (NFATc1), which plays a critical role in calcium/calcineurin signaling. Taken together, our results demonstrate that CaSR is expressed in hAFMSCs, positively correlates with osteogenic markers, and is activated by R-568. Notably, downregulation of CaSR by RNA interference supports the conclusion that CaSR activation plays a central role in hAFMSC osteogenesis. Thus, this study provides significant information on the mechanisms of hAFMSC osteogenesis, which could provide additional molecular basis for the use of calcimimetics in bone regenerative medicine.

PRKDC mutations associated with immunodeficiency, granuloma and aire-dependent autoimmunity

We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA DSB repair and V(D)J recombination. Circulating T cells had a skewed cytokine response typical of Th1 and Th2 profiles. Moreover, mutated DNA-PKcs failed to promote AIRE-dependent transcription of peripheral tissue antigens in vitro .T he latter defect correlated in vivo, with the production of antiCalcium Sensing Receptor (anti-CaSR) autoantibodies, which are usually found in AIRE-deficient patients. Conclusion Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoantibodies and these findings highlight the essential role of DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human. Disclosure of interest None declared.

The expression of calcium-sensing receptor in hyperglacemia induced macroangiopathy

Objective To explore the changes of calcium-sensing receptor (CaSR) in hyperglacemia induced macroangiopathy and the mechanisms.Methods SD rats were randomly divided into control group (Con,n =9) and diabetic group (DM,n =9),diabetic rats were made with streptozotocin (STZ) and high-fat diet.Hydroxyproline level in thoracic aorta was measured via ChoramnieT oxidation method.Message RNA level of CaSR was detected via real-time quantitative polymerase chain reaction (Real-time PCR),and protein level of CaSR and B cell lymphoma/leukemia-2 associated X protein (bax) was detected by Western blotting.Cell apoptosis was analyzed through TdT-mediated dUTP nick end labeling (TUNEL) assay.Results The hydroxyproline [(1.380 ± 0.030) mg/L],the CaSR mRNA (3.960 ± 0.110),CaSR protein 1 (2.600 ± 0.170) and bax protein level (3.201 ±0.177) in diabetic group were higher than control group [(1.053 ± 0.041) mg/L,(1.003 ± 0.015),(1.000 ± 0.032),(1.000 ± 0.084)] (P ＜ 0.05),the apoptotic ratic increased by about 30％ in diabetic group (P ＜ 0.05).Conclusion The CaSR mRNA and CaSR protern in diabetic group were higher than control group,CaSR might participate in the pathogenesis and development of hyperglycemia induced macroangiopathy through promoting bax related apoptosis pathway and initiating programmed cell death. Key words: Calcium sensing receptor; Diabetes; Macroangiopathy ; Apoptosis

Which outcome in chronic kidney disease-mineral and bone disorder patients?

Dear Editor, Secondary hyperparathyroidism (SHPT) is one the major complications in dialysis patients. Hyperphosphatemia, hypocalcemia, low serum vitamin D levels are greatly involved into the physiopathology of SHPT in chronic kidney disease (CKD), in which the down-regulation of parathyroid Vitamin D Receptor (VDR) and Calcium Sensing Receptor (CaSR) represent a critical step. Moreover, new data demonstrate that the Fibroblast Growth Factor 23 (FGF-23) plays an important role in the regulation of calcium-phosphate-vitamin D axis in CKD. Thus, new insights into the pathogenesis of SHPT give the possibility to have different treatments of this condition in CKD. SHPT causes both bone (osteitis fibrosa) and extra-skeletal disease (cardiovascular and soft-tissue calcifications, altered erythropoiesis, endocrine dysfunctions) (1). Clinical targets for serum phosphate (P), calcium (Ca), and parathyroid hormone (PTH) levels have been elucidated by the Kidney Dialysis Outcomes Quality Initiative (K/DOQI) Working Group (2). Since alterations in mineral and bone metabolism have classically been named renal osteodystrophy and classified based on bone biopsy, recently, KDIGO (Kidney Disease: Improving Global Outcomes) proposed a new definition for this disease: the CKD-Mineral and Bone Disorder (CKD-MBD) (3). In addition to hypocalcemia and hyperphosphatemia, CKD-MBD is characterized by abnormalities of bone volume, turnover, mineralization, and growth as well as vascular calcification (VC). In fact, VC seems to link abnormalities in serum Ca and P levels and the enhanced mortality risk associated with CKD-MBD. Impressively, VC starts early in CKD and patients with CKD-3, -4 and -5 not undergoing hemodialysis present a significant amount of calcium deposition in the coronary arteries (40-80%). During the last decade, many efforts have been made to try to decrease VC progression. The control of serum PTH levels and calcium- and phosphate-load, using aluminum- and calcium phosphate binders, more selective vitamin D receptors activators, and cinacalcet clearly represent our current pharmacological tools to improve quality of life and reduce mortality in CKD (4). In their manuscript, Rostami et al. (5) showed quality of life in dialysis patients affected by CKD-MBD in Iran. Interestingly, quality of like associated with serum Ca levels, serum calcium-phosphate product levels, and serum PTH levels. Furthermore, hospitalizations were associated with serum P levels. Thus, management of CKD-MBD included phosphate binders (both calcium-based and calcium-free), vitamin D, and cinacalcet, even if treatment of this dramatic complication of CKD remains suboptimal for most patients.

Neonatal severe hyperparathyroidism due to compound heterozygous mutation of calcium sensing receptor (CaSR) gene presenting as encephalopathy.

The authors report a 14-d-old neonate who presented with lethargy, polyuria and dehydration and was found to have severe hypercalcemia with hyperparathyroidism. This neonate was treated with saline hydration, diuresis and injection pamidronate. Genetic analysis revealed a compound heterozygous mutation of CaSR.

NKCC2 activity is inhibited upon the expression of the gain‐of‐function CASR mutant A843E in renal cells (892.23)

The mutation gain‐of‐function A843E of the Calcium Sensing Receptor (CASR) causes a Bartter syndrome type V. Patients carrying this CASR variant showed a remarkable reduced renal NaCl reabsorption in Thick Ascendent Limb (TAL) resulting in renal loss of NaCl in the absence of mutations in renal Na+ and Cl‐ ion transporters. The molecular mechanisms underlying this clinical phenotype are still unknown. Indeed, we investigated, in LLC‐PK1 epithelial renal cells, the possible functional cross‐talk of CASR‐A843E mutant with NKCC2, the main transporter involved in the NaCl reabsorption in the TAL. The steady‐state NKCC2 phosphorylation and activity were inhibited by about 60% in cells transfected with CASR‐A843E mutant compared to CASR WT‐transfected cells, used as control. Of note, the low‐chloride‐dependent NKCC2 activation was also strongly inhibited upon the expression of CASR‐A843E mutant. Interestingly the maximal activation of CASR WT mimicked the effect of CASR‐A843E on NKCC2 regulation. The expression of the CASR mutant did not alter the apical localization of NKCC2 in LLC‐PK1 cells, suggesting that the CASR‐A843E affected intracellular pathway/s modulating NKCC2 activity rather than NKCC2 intracellular trafficking. Our findings opened new avenues not only to gain more insight in the role of CASR in the physiopathology of the kidney, but also to conceive novel therapeutic strategies for the treatment of the Bartter syndrome type V.Grant Funding Source: Supported by PRIN 2009 n# 2009J53ALK to MS

Role of Receptor Activity Modifying Protein 1 in Function of the Calcium Sensing Receptor in the Human TT Thyroid Carcinoma Cell Line

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca2+ and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.

Acidosis Sensing Receptor GPR65 Correlates with Anti-Apoptotic Bcl-2 Family Member Expression in CLL Cells: Potential Implications for the CLL Microenvironment.

The tumor microenvironment is generally an acidic environment, yet the effect of extracellular acidosis on chronic lymphocytic leukemia (CLL) is not well established. Here we are the first to report that the extracellular acid sensing G-protein coupled receptor, GPR65, is expressed in primary CLL cells where its level correlate strongly with anti-apoptotic Bcl-2 family member levels. GPR65 expression is found normally within the lymphoid lineage and has not been previously reported in CLL. We demonstrate a wide range of GPR65 mRNA expression among CLL 87 patient samples. The correlation between GPR65 mRNA levels and Bcl-2 mRNA levels is particularly strong (r=0.8063, p= <0.001). The correlation extends to other anti-apoptotic Bcl-2 family members, Mcl-1 (r=0.4847, p=0.0010) and Bcl-xl (r=0.3411, p=0.0252), although at lower levels of significance. No correlation is detected between GPR65 and levels of the pro-apoptotic proteins BIM, PUMA or NOXA. GPR65 expression also correlates with the favorable prognostic marker of 13q deletion. The present findings suggest the acid sensing receptor GPR65 may be of significance to allow CLL tolerance of extracellular acidosis. The correlation of GPR65 with Bcl-2 suggests a novel cytoprotective mechanism that enables CLL cell adaptation to acidic extracellular conditions. These findings suggest the potential value of targeting GPR65 therapeutically.

Selective Imaging of Quorum Sensing Receptors in Bacteria Using Fluorescent Au Nanocluster Probes Surface Functionalized with Signal Molecules

Fluorescent ultrasmall gold clusters decorated with bacterial quorum sensing signal molecules, acyl homoserine lactone, are synthesized. These fluorescent probes are found to have emission in the near-infrared spectral region advantageous for bioimaging. Imaging studies using different strains of bacteria with and without acyl homoserine lactone receptors with the aid of confocal microscopy have shown that the probe interacts preferentially with cells possessing these receptors. This indicates that, with appropriate surface functionalization, the Au clusters can be used for receptor specific detection with enhanced selectivity.

Structural Characterization of Native Autoinducing Peptides and Abiotic Analogues Reveals Key Features Essential for Activation and Inhibition of an AgrC Quorum Sensing Receptor in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen that uses quorum sensing (QS) to control virulence. Its QS system is regulated by macrocyclic peptide signals (or autoinducing peptides (AIPs)) and their cognate transmembrane receptors (AgrCs). Four different specificity groups of S. aureus have been identified to date (groups I-IV), each of which uses a different AIP:AgrC pair. Non-native ligands capable of intercepting AIP:AgrC binding, and thereby QS, in S. aureus have attracted considerable interest as chemical tools to study QS pathways and as possible antivirulence strategies for the treatment of infection. We recently reported a set of analogues of the group-III AIP that are capable of strongly modulating the activity of all four AgrC receptors. Critical to the further development of such ligands is a detailed understanding of the structural features of both native AIPs and non-native analogues that are essential for activity. Herein, we report the first three-dimensional structural analysis of the known native AIP signals (AIPs-I-IV) and several AIP-III analogues with varied biological activities using NMR spectroscopy. Integration of these NMR studies with the known agonism and antagonism profiles of these peptides in AgrC-III revealed two key structural elements that control AIP-III (and non-native peptide) activity: (1) a tri-residue hydrophobic "knob" essential for both activation and inhibition and (2) a fourth anchor point on the exocyclic tail needed for receptor activation. These results provide strong structural support for a mechanism of AIP-mediated AgrC activation and inhibition in S. aureus , and should facilitate the design of new AgrC ligands with enhanced activities (as agonists or antagonists) and simplified chemical structures.

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

Calcium Sensing Receptor is expressed on/in osteocyte-like MLOY4 culture and modulated by strontium ranelate

Calcium Sensing Receptor is expressed on/in osteocyte-like MLOY4 culture and modulated by strontium ranelate

592 Are Epigenetic Mechanisms Responsible for Silencing the Calcium Sensing Receptor in Colorectal Cancer?

592 Are Epigenetic Mechanisms Responsible for Silencing the Calcium Sensing Receptor in Colorectal Cancer?

Zinc Sensing Receptor Signaling, Mediated by GPR39, Reduces Butyrate-Induced Cell Death in HT29 Colonocytes via Upregulation of Clusterin

Zinc enhances epithelial proliferation, protects the digestive epithelial layer and has profound antiulcerative and antidiarrheal roles in the colon. Despite the clinical significance of this ion, the mechanisms linking zinc to these cellular processes are poorly understood. We have previously identified an extracellular Zn2+ sensing G-protein coupled receptor (ZnR) that activates Ca2+ signaling in colonocytes, but its molecular identity as well as its effects on colonocytes' survival remained elusive. Here, we show that Zn2+, by activation of the ZnR, protects HT29 colonocytes from butyrate induced cell death. Silencing of the G-protein coupled receptor GPR39 expression abolished ZnR-dependent Ca2+ release and Zn2+-dependent survival of butyrate-treated colonocytes. Importantly, GPR39 also mediated ZnR-dependent upregulation of Na+/H+ exchange activity as this activity was found in native colon tissue but not in tissue obtained from GPR39 knock-out mice. Although ZnR-dependent upregulation of Na+/H+ exchange reduced the cellular acid load induced by butyrate, it did not rescue HT29 cells from butyrate induced cell death. ZnR/GPR39 activation however, increased the expression of the anti-apoptotic protein clusterin in butyrate-treated cells. Furthermore, silencing of clusterin abolished the Zn2+-dependent survival of HT29 cells. Altogether, our results demonstrate that extracellular Zn2+, acting through ZnR, regulates intracellular pH and clusterin expression thereby enhancing survival of HT29 colonocytes. Moreover, we identify GPR39 as the molecular moiety of ZnR in HT29 and native colonocytes.

Determination of Calcium Sensing Receptor in the Scales of Goldfish and Induction of Its mRNA Expression by Acceleration Loading

Determination of Calcium Sensing Receptor in the Scales of Goldfish and Induction of Its mRNA Expression by Acceleration Loading

ChemInform Abstract: Design and Synthesis of Cyclic Sulfonamides and Sulfamates as New Calcium Sensing Receptor Agonists.

AbstractThe synthetic strategy used is based on imposing conformational restrictions to the first calcimimetic family previously synthesized.

Expression of calcium sensing receptor and receptor activity modifying proteins in cells involved in calcium homeostasis and fluorescence resonance energy transfer based stoichiometric analysis of their interactions

Event Abstract Back to Event Expression of calcium sensing receptor and receptor activity modifying proteins in cells involved in calcium homeostasis and fluorescence resonance energy transfer based stoichiometric analysis of their interactions AJ Desai1*, DJ Roberts1, GO Richards1 and TM Skerry1 1 University of Sheffield, Department of Human Metabolism, United Kingdom {BR}The G-protein coupled receptor known as the Calcium Sensing Receptor (CaSR) is known to play an important role in calcium homeostasis by sensing changes in extracellular calcium and modulating secretion of calcitropic hormones. A recent study revealed that cell surface expression of the CaSR requires its association with single transmembrane accessory protein called Receptor Activity Modifying Protein (RAMP), specifically either RAMP1 or 3.{BR}We hypothesize that efficiency of interaction and co-localization of RAMPs with CaSR could be determined using stoichiometric Fluorescence Resonance Energy Transfer (FRET) analysis. {BR}In transfected COS7 cells, CaSR+RAMP1/3 complexes are highly co-localized around the peri-nuclear region indicative of co-localization in the endoplasmic reticulum and the Golgi bodies and also in regions of the cell membrane. The CaSR+RAMP3 FRET complex is ~25% more efficient than CaSR+RAMP1 FRET complex. Furthermore, the fraction of RAMP3 involved in FRET is ~18.8% more than RAMP1 whereas the fraction of CaSR involved in FRET with RAMP3 is ~33% more than with RAMP1. {BR}To determine whether RAMP interactions with CaSR in cells involved in calcium homeostasis had roles beyond trafficking, we explored their expression in thyroid and bone cell lines.{BR}We detected the expression of mRNA and protein for CaSR, RAMP1 and 2 in thyroid medullary carcinoma cell line (TT cells) using quantitative PCR, immunocytochemistry and western blotting. However, we could not detect expression of CaSR and RAMP3 mRNA transcripts in pre-osteoblastic MG63, SAOS-2 and TE85 cell lines using quantitative PCR. A high dose of extracellular calcium and differentiation of these cells into mature osteoblasts did not induce expression of CaSR or RAMP3. This suggests that osteosarcoma cell lines are not ideal models of osteoblasts to study CaSR expression and function.{BR}The novel finding of the current study is that it provides a stoichiometric insight into CaSR-RAMP interaction and also indicates that CaSR interacts more efficiently with RAMP3 efficiently than RAMP1. This data is a step forward from the existing knowledge of CaSR-RAMP interaction. The increased understanding of their interaction will clarify their role in physiology and patho-physiology. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Poster Topic: Abstracts Citation: Desai A, Roberts D, Richards G and Skerry T (2011). Expression of calcium sensing receptor and receptor activity modifying proteins in cells involved in calcium homeostasis and fluorescence resonance energy transfer based stoichiometric analysis of their interactions. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00013 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Mr. AJ Desai, University of Sheffield, Department of Human Metabolism, United Kingdom, a.desai@sheffield.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers AJ Desai DJ Roberts GO Richards TM Skerry Google AJ Desai DJ Roberts GO Richards TM Skerry Google Scholar AJ Desai DJ Roberts GO Richards TM Skerry PubMed AJ Desai DJ Roberts GO Richards TM Skerry Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche

AbstractAbstract 399The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. Disclosures:No relevant conflicts of interest to declare.