title: Nitric oxide enhances osteoclastogenesis possibly by mediating cell fusion.
abstract: Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. Here, we investigate how nitric oxide (NO) affects osteoclastogenesis. Time lapse photomicrography, using the fluorescent NO indicator dye, 4,5-diaminofluorescein diacetate, revealed an intense NO signal in pre-osteoclasts preceding cell fusion. Osteoclastogenesis in RAW264.7 cells increased when exposed to the NO synthase inhibitor, L-NMMA (0.25 microM), for the initial 48 h. In contrast, pre-osteoclast fusion decreased when RAW264.7 cells were exposed to L-NMMA from 48 to 96 h. Both NO synthase inhibitors, L-NMMA and L-NAME, decreased osteoclast formation during this time period. The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations (25-200 ng/ml) of sRANKL suggesting signaling cross talk. NO donors increased osteoclast formation in a dose-dependent manner, with greatest stimulation at 15 microM NOC-12 (2.3 fold) and 5 microM NOC-18 (2.4 fold). Measuring nitrite (NO end product) daily from culture media of RAW264.7 cells undergoing osteoclastogenesis revealed that an increase in NO production coincided with the fusion of pre-osteoclasts (day 4). Inhibiting fusion by plating cells on polystyrene dishes pre-coated with poly-(L-lysine) decreased both osteoclast formation and NO production. To address if NO mediates fusion through the actin cytoskeleton, actin free barbed ends were measured. 0.25 microM L-NMMA decreased, while 15 microM NOC-12 and 5 microM NOC-18 increased actin free barbed ends. We hypothesize that while NO initially negatively regulates pre-osteoclast differentiation; it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling.
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
authors.forename: Dorrin
authors.initials: D
authors.lastname: Nilforoushan
authors.forename: Azza
authors.initials: A
authors.lastname: Gramoun
authors.forename: Michael
authors.initials: M
authors.lastname: Glogauer
authors.forename: Morris F
authors.initials: MF
authors.lastname: Manolson
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010784:Photomicrography; D011107:Polylysine / Q000494:pharmacology; D011817:Rabbits; D000071681:Tartrate-Resistant Acid Phosphatase; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C093285:NOC 18; D020030:Nitric Oxide Donors; D009573:Nitrites; D009603:Nitroso Compounds; D011107:Polylysine; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase