Said Kamel

The Calcium-sensing Receptor Is Involved in Strontium Ranelate-induced Osteoclast Apoptosis NEW INSIGHTS INTO THE ASSOCIATED SIGNALING PATHWAYS

Strontium ranelate exerts both an anti-catabolic and an anabolic effect on bone cells. To further investigate the molecular mechanism whereby strontium ranelate inhibits bone resorption, we focused our attention on the effects of strontium ranelate on osteoclast apoptosis and on the underlying mechanism(s). Using primary mature rabbit osteoclasts, we demonstrated that strontium \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{Sr}_{\mathrm{o}}^{2+})\) \end{document} dose-dependently stimulates the apoptosis of mature osteoclasts. As shown previously for calcium \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{Ca}_{\mathrm{o}}^{2+})\) \end{document}, the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document}-induced effect on mature osteoclasts is mediated by the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}-sensing receptor, CaR, which in turn stimulates a phospholipase C-dependent signaling pathway and nuclear translocation of NF-κB. Unlike \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}, however, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document}-induced osteoclast apoptosis was shown to depend on PKCβII activation and to be independent of inositol 1,4,5-trisphosphate action. As a consequence of these differences in their intracellular signaling pathways, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document} in combination were shown to exert a greater effect on mature osteoclast apoptosis than did either divalent cation by itself. Altogether, our results show that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} acts through the CaR and induces osteoclast apoptosis through a signaling pathway similar to but different in certain respects from that of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}. This difference in the respective signaling cascades enables \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} to potentiate \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}-induced osteoclast apoptosis and vice versa. In this manner, it is conceivable that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document} act together to inhibit bone resorption in strontium ranelate-treated patients.

The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis

Intracellular transduction pathways that are dependent on activation of the CaR by Cao2+ have been studied extensively in parathyroid and other cell types, and include cytosolic calcium, phospholipases C, A2, and D, protein kinase C isoforms and the cAMP/protein kinase A system. In this study, using bone marrow cells isolated from CaR−/− mice as well as DN‐CaR‐transfected RAW 264.7 cells, we provide evidence that expression of the CaR plays an important role in osteoclast differentiation. We also establish that activation of the CaR and resultant stimulation of PLC are involved in high Cao2+‐induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Cao2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF‐κB in a CaR‐ and PLC‐dependent manner. In summary, our data suggest that stimulation of the CaR may play a pivotal role in the control of both osteoclast differentiation and apoptosis in the systems studied here through a signaling pathway involving activation of the CaR, phospholipase C, and NF‐κB.— Mentaverri, R., Yano, S., Chattopadhyay, N., Petit, L., Kifor, O., Kamel, S., Terwilliger, E. F., Brazier, M., Brown, E. M. The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis. FASEB J. 20, E1945‐E1954 (2006)

The calcimimetic R-568 retards uremia-enhanced vascular calcification and atherosclerosis in apolipoprotein E deficient (apoE―/―) mice

OBJECTIVE Secondary hyperparathyroidism of chronic kidney disease promotes vascular calcification. Calcimimetics reduce serum parathyroid hormone, calcium (Ca), and phosphorus by calcium-sensing receptor (CaR) activation. Here we examined possible effects of the calcimimetic R-568 (R-568) on the progression of aortic calcification and atherosclerosis in apoE(-/-) mice with chronic renal failure (CRF) and the potential implication of aortic smooth muscle cell CaR. METHODS AND RESULTS ApoE(-/-) mice were assigned to 3 CRF groups and 1 non-CRF group receiving daily gavage with R-568, calcitriol, or vehicle. Serum Ca and phosphorus and parathyroid gland volume of CRF mice were decreased by R-568, whereas elevated serum FGF23 and total cholesterol remained unchanged. Both aortic plaque and non-plaque calcification was lower in R-568 mice, and so was atherosclerotic plaque area fraction. In vitro, R-568 induced a decrease in smooth muscle cell calcification when cultured in high phosphate medium. This decrease was abolished in CaR-SiRNA-transfected cells. CONCLUSIONS The calcimimetic R-568 delayed the progression of both aortic calcification and atherosclerosis in uremic apoE(-/-) mice. This effect was mediated via a better control of hyperparathyroidism including serum Ca and phosphorus. Direct vascular CaR activation also could have played a role in the observed effects.

Low-Gradient, Low-Flow Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction: Characteristics, Outcome, and Implications for Surgery

BACKGROUND Severe low-gradient, low-flow (LG/LF) aortic stenosis with preserved left ventricular ejection fraction (EF) has been described as a more advanced form of aortic stenosis. However, the natural history and need for surgery in patients with LG/LF aortic stenosis remain subjects of intense debate. OBJECTIVES We sought to investigate the outcome of LG/LF aortic stenosis in comparison with moderate aortic stenosis and with high-gradient (HG) aortic stenosis in a real-world study, in the context of routine practice. METHODS This analysis included 809 patients (ages 75 ± 12 years) diagnosed with aortic stenosis and preserved EF (≥50%). Patients were divided into 4 groups: mild-to-moderate aortic stenosis; HG aortic stenosis; LG/LF aortic stenosis; and low-gradient, normal-flow (LG/NF) aortic stenosis. RESULTS Compared with mild-to-moderate aortic stenosis patients, LG/LF aortic stenosis patients had smaller valve areas and stroke volumes, higher mean gradients, and comparable degrees of ventricular hypertrophy. Under medical management (22.8 months; range 7 to 53 months), compared with mild-to-moderate aortic stenosis patients, HG aortic stenosis patients were at higher risk of death (adjusted hazard ratio [HR]: 1.47; 95% confidence interval [CI]: 1.03 to 2.07), whereas LG/LF aortic stenosis patients did not have an excess mortality risk (adjusted HR: 0.88; 95% CI: 0.53 to 1.48). During the entire (39.0 months; range 11 to 69 months) follow-up (with medical and surgical management), the mortality risk associated with LG/LF aortic stenosis was close to that of mild-to-moderate aortic stenosis (adjusted HR: 0.96; 95% CI: 0.58 to 1.53), whereas the excess risk of death associated with HG aortic stenosis was confirmed (adjusted HR: 1.74; 95% CI: 1.27 to 2.39). The benefit associated with aortic valve replacement was confined to the HG aortic stenosis group (adjusted HR: 0.29; 95% CI: 0.18 to 0.46) and was not observed for LG/LF aortic stenosis (adjusted HR: 0.75; 95% CI: 0.14 to 4.05). CONCLUSIONS In this study, the outcome of severe LG/LF aortic stenosis with preserved EF was similar to that of mild-to-moderate aortic stenosis and was not favorably influenced by aortic surgery. Further research is needed to better understand the natural history and the progression of LG/LF aortic stenosis.

Effects on bone mineral density of calcium and vitamin D supplementation in elderly women with vitamin D deficiency.

OBJECTIVE Calcium and vitamin D deficiency is common in older individuals, particularly those who live in nursing homes, and increases the risk of osteoporosis and fractures. METHODS We conducted a randomized double-blind placebo-controlled study of combined supplementation with 500 mg of elemental calcium, as carbonate, and 400 IU of vitamin D bid for 12 months in women older than 65 years of age with vitamin D deficiency, defined as serum 25(OH)D concentrations </=12 ng/ml. RESULTS Mean patient age was 75 +/- 7 years, and median daily dietary intakes of calcium and vitamin D were 697 mg and 66.8 IU in the supplemented group (n = 95) and 671 mg and 61.8 IU in the placebo group (n = 97). The median serum 25(OH)D level was 7.0 ng/ml in both groups, and the medial intact parathyroid hormone (PTHi) levels were 49 and 48 pg/ml in the supplemented and placebo groups, respectively. The median increase in serum 25(OH)D was 22.0 ng/ml in the supplemented group and 4 ng/ml in the placebo group (P < 0.0001), and the median PTHi decrease was 17 and 5 pg/ml, respectively (P < 0.0001). The median bone mineral density increase was significantly greater in the supplemented group than in the placebo group: +2.98% vs. -0.21% at L2-L4 (P = 0.0009), +1.19% and -0.83% at the femoral neck (P = 0.015), +0.86% and -0.56% at the trochanter (P = 0.015), and +0.99% and +0.11% for the whole body (P = 0.01). Similarly, the median decrease in the main bone markers was significantly greater in the treated group than in the placebo group: -1.35 microg/l vs. +0.50 microg/l for bone alkaline phosphatase (P = 0.008), -16.6 nmol/mmol creatinine vs. -2.3 nmol/mmol creatinine for urinary type I amino-terminal telopeptide (P = 0.001), and -896 pmol/l vs. -201 pmol/l for serum type I carboxy-terminal telopeptide (P = 0.003). We found no significant differences between the two groups for serum calcium, although urinary calcium excretion changed more in the supplemented group than in the placebo group. In conclusion, bone mass in older women with vitamin D deficiency increases significantly at the lumbar spine, femur, trochanter, and whole body after calcium and vitamin D supplementation for 1 year, and concomitantly bone markers improved as vitamin D levels returned to normal.

High extracellular inorganic phosphate concentration inhibits RANK–RANKL signaling in osteoclast-like cells†

In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an “osteoclastic” phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte–macrophage precursor cells to differentiate into osteoclast‐like cells in the presence of receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA‐binding activity of the transcription factors AP‐1 and NF‐κB. Increase in extracellular Pi concentration (1.5–4.5 mM) dose‐dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M‐CSF. Pi was found to specifically inhibit the RANKL‐induced JNK and Akt activation, while RANKL‐induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL‐stimulated DNA‐binding activity of NF‐κB. The effects of Pi on osteoclast differentiation and DNA‐binding activity of NF‐κB were prevented by Foscarnet®, a sodium–phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK–RANKL signaling. J. Cell. Physiol. 215: 47–54, 2008.

Extracellular signal-regulated kinases 1 and 2 and TRPC1 channels are required for calcium-sensing receptor-stimulated MCF-7 breast cancer cell proliferation.

The calcium-sensing receptor (CaR), is a G protein-dependent receptor that responds to increments in extracellular Ca²⁺ ([Ca²⁺]_o). We previously reported that an increase in [Ca²⁺]_o induced a release of intracellular calcium and Ca²⁺ entry via store operated channels (SOCs). We also demonstrated that MCF-7 cells express Transient Receptor Potential canonical 1 (TRPC1) channels. Herein, we investigated CaR intracellular signaling pathways and examined the role of TRPC1 in CaR-induced cell proliferation, through the extracellular signal-regulated Kinases 1 & 2 (ERK1/2) pathways. Treatment by [Ca²⁺]_o increased both MCF-7 cell proliferation and TRPC1 expression. Both the [Ca²⁺]_o proliferative effect and TRPC1 protein levels were abolished by the ERK1/2 inhibitors. Moreover, [Ca²⁺]_o failed to increase cell proliferation either in the presence of CaR or TRPC1 siRNAs. Both [Ca²⁺]_o and the selective CaR activator spermine, elicited time and dose-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation was almost completely inhibited by treatment with the phospholipase C and the protein kinase C inhibitors. Treatment with 2-aminoethoxydiphenyl borate (2-APB), and SKF-96365 or by siTRPC1 diminished both [Ca²⁺]_o- and spermine-stimulated ERK1/2 phosphorylation. Moreover, down-regulation of TRPC1 by siRNA reduced the Ca²⁺ entry induced by CaR activation. We conclude that the CaR activates ERK1/2 via a PLC/PKC-dependent pathway. Moreover, TRPC1 is required for the ERK1/2 phosphorylation, Ca²⁺ entry and the CaR-proliferative effect.

Strontium Ranelate Decreases Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclastic Differentiation In Vitro: Involvement of the Calcium-Sensing Receptor

Strontium ranelate exerts both an anticatabolic and an anabolic effect on bone cells. To further investigate the mechanism by which strontium ranelate inhibits bone resorption, the effects of varying concentrations of Sro2+ on osteoclastic differentiation were studied using RAW 264.7 cells and peripheral blood monocytic cells (PBMCs). We report that increasing concentrations of Sro2+ down-regulate osteoclastic differentiation and tartrate-resistant acid phosphatase activity, leading to inhibition of bone resorption (−48% when PBMCs were cultured for 14 days in the presence of 2 mM Sro2+). Using a dominant-negative form of the calcium-sensing receptor (CaR) and a small interfering RNA approach, we provide evidences that the inhibition of osteoclast differentiation by Sro2+ is mediated by stimulation of the CaR. Moreover, our results suggest that the effects of Sro2+ on osteoclasts are, at least in part, mediated by inhibition of the receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear translocation of nuclear factor-κB and activator protein-1 in the early stages of osteoclastic differentiation. In conclusion, our data indicate that Sr2+ directly inhibits the formation of mature osteoclasts through down-regulation of RANKL-induced osteoclast differentiation and decreases osteoclast differentiation through the activation of the CaR.

Agonists and Allosteric Modulators of the Calcium-Sensing Receptor and Their Therapeutic Applications

The calcium-sensing receptor (CaR) belongs to the G protein-coupled receptor superfamily, with a characteristic structure consisting of seven transmembrane helices, an intracellular C-terminal and an extracellular N terminal domain. The primary physiological function of the CaR is the maintenance of constant blood Ca2+ levels, as a result of its ability to sense very small changes in extracellular Ca2+ (Ca2+o). Nevertheless, in addition to being expressed in tissues involved in Ca2+o homeostasis, the CaR is also expressed in tissues not involved in mineral homeostasis, suggestive of additional physiological functions. Numerous agonists and modulators of the CaR are now known in addition to Ca2+o, including various divalent and trivalent cations, aromatic l-amino acids, polyamines, and aminoglycoside antibiotics. The signaling of the CaR is also regulated by extracellular pH and ionic strength. The activated CaR couples mainly to the phospholipase Cβ and extracellular signal-regulated kinase 1/2 signaling pathways, and it decreases intracellular cAMP levels, leading to various physiological effects. The recent identification of synthetic allosteric modulators of the CaR has opened up a new field of research possibilities. Calcimimetics and calcilytics, which increase and decrease agonist signaling via the CaR, respectively, may facilitate the manipulation of the CaR and thus aid in further investigations of its precise signaling. These allosteric modulators, as well as strontium, have been demonstrated to have therapeutic potential for the treatment of disorders involving the CaR. This review discusses the various agonists and modulators of the CaR, differences in their binding and signaling, and their roles as therapeutics in various diseases.

The circulating soluble TRAIL is a negative marker for inflammation inversely associated with the mortality risk in chronic kidney disease patients

BACKGROUND Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and an inadequate inflammatory response which may account for the high morbidity and mortality observed in this population. In vitro and preclinical evidence suggests that the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) might be involved in both the atherosclerosis pathway and modulation of the inflammatory response. The aim of the present study was thus to (i) determine serum levels of soluble TRAIL (sTRAIL) in a cohort of CKD patients, (ii) assess the relationship between sTRAIL and other inflammatory biomarkers (C-reactive protein and albumin) and (iii) evaluate the association between serum sTRAIL levels and the mortality risk. METHODS One hundred and thirty patients (mean +/- SD age: 67 +/- 12; 62% males; 8% at CKD stage 2, 26% at stage 3, 27% at stage 4, 8% at stage 5 and 31% at stage 5D) were assayed for sTRAIL and the selected biochemical parameters and then prospectively monitored for mortality. RESULTS CKD stage 5D patients had significantly lower serum sTRAIL levels (median: 46 pg/ml) than patients at CKD stages 2 and 3 (median: 62 pg/ml) or stages 4 and 5 (median: 71 pg/ml). There was no correlation between serum sTRAIL and the estimated glomerular filtration rate (GFR) (r(2) = 0.017, P = 0.22) in pre-dialysis patients. In a multivariate regression analysis, the body mass index (beta = 1.48, P = 0.001) and the serum C-reactive protein (CRP) level (beta = -8.841, P < 0.0001) were independently associated with serum sTRAIL. During follow-up (mean: 772 +/- 286 days), 36 patients died (19 from cardiovascular events, 8 from infectious events and 9 from other causes). The lowest sTRAIL levels (first tertile) were associated with the worst all-cause survival (P = 0.010). Cox regression analyses (with non-cumulative models including age, albumin and CRP as covariates) confirmed the low serum sTRAIL level (first tertile) as an independent predictor of all-cause mortality. CONCLUSIONS Circulating sTRAIL is a negative marker for inflammation and is inversely associated with the mortality risk in CKD patients. Further studies are needed to better understand the role of sTRAIL as an inflammatory marker and to confirm its protective role in the CKD population.