Journal: The Journal of Biological Chemistry

Article Title: TRPV-5 Mediates a Receptor Activator of NF-?B (RANK) Ligand-induced Increase in Cytosolic Ca 2+ in Human Osteoclasts and Down-regulates Bone Resorption * ^♦

doi: 10.1074/jbc.M109.075234

Figure Lengend Snippet: TRPV-5 siRNA inhibited RANKL-induced increase in [Ca2+]i and enhanced bone resorption. CBMs were cultured for 21 days in the presence of macrophage-CSF and RANKL. After 17 days, mature osteoclasts were transfected with an irrelevant control siRNA (CTL−), transfected with an anti-TRPV-5 siRNA, or left untreated (NT). A, quantitative PCR analysis of TRPV-5 expression in cells transfected either with a non-relevant control siRNA or with an anti-TRPV-5 siRNA. Quantitative PCR was performed on RNA extracted 6, 8, 18, 24, or 48 h after siRNA transfection. The results of four independent experiments were computed and reported graphically. *, p < 0.5, **, p < 0.01 versus time 0. B, Western blot (WB) analysis of TRPV-5 expression was performed with proteins extracted 24 h after siRNA transfection and with antibodies directed against TRPV-5 or actin. The graph shows the ratio of averaged TRPV-5 over actin optical densities obtained in Western blots in four independent experiments. C, the cells were transfected with an irrelevant control siRNA (dashed line) or with an anti-TRPV-5 siRNA (solid line) and were loaded with Fura2-AM. They were then stimulated with RANKL (100 ng/ml), and the intracellular [Ca2+] was recorded. One representative graph of three experiments is shown. D, average increase in [Ca2+]i after RANKL stimulation in cells transfected with an irrelevant control siRNA or an anti-TRPV-5 siRNA. Results are from three experiments with at least eight osteoclasts analyzed per field. E, CBMs were settled on bovine bone slices and cultured for 21 days. After 17 days, cells were transfected as described above. Bone resorption was evaluated at day 22, and the percentage of area resorbed was computed. The graph shows the average results from three independent experiments. *, p < 0.5, **, p < 0.01 versus control (CTL-).

Article Snippet: Recombinant human macrophage-CSF and recombinant human granulocyte-macrophage-CSF were purchased from R&D Systems (Minneapolis, MN); goat polyclonal antibodies against calcitonin receptor (CTR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal antibodies against TRPV-5 were from Abcam (Cambridge, MA), and rabbit polyclonal antibodies were from Upstate Biotech Millipore (Billerica, MA); rabbit polyclonal antibodies against L-type and T-type Ca 2+ channels were purchased from Sigma-Aldrich; those against R-type channels were from Novus Biologicals (Littleton, CO); and rabbit polyclonal antibodies against V-ATPase were purchased from Chemicon (Temecula, CA).

Techniques: Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot