title: A possible role for nitric oxide in osteoclastogenesis associated with cholesteatoma.
abstract: OBJECTIVE
  This study was designed to investigate the potential role of nitric oxide in cholesteatoma-induced bone resorption, in vitro and in vivo.
  
  
  BACKGROUND
  Cholesteatoma is a disease of inflammatory bone resorption in the middle ear leading to hearing loss and vestibular dysfunction. Inappropriate activation of osteoclasts causes the morbidity associated with this disease. Previous studies suggest nitric oxide may be an important mediator of osteoclast function.
  
  
  METHODS
  A murine model of cholesteatoma induced bone resorption was used to demonstrate nitric oxide synthase (NOS) gene expression and the effect of a NOS inhibitor. An in vitro osteoclast culture method was used to demonstrate the effect of nitric oxide on isolated osteoclasts. Osteoclast development was assayed by counting the number of mature osteoclasts; activity was assayed by measuring the amount of resorbed bone.
  
  
  RESULTS
  Quantitative reverse transcriptase-polymerase chain reaction results demonstrated the temporal expression of all three NOS isoforms in vivo. NOS I demonstrated very low levels of expressions throughout the duration of the study with no change in expression in response to keratin implant. Similarly, NOS III also demonstrated low levels of expression and no change in response to keratin. NOS II was highly upregulated in response to keratin throughout the duration of the study. In vitro, pharmacological nitric oxide donors--sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine--dose-dependently stimulated osteoclast resorption. Alone, interferon gamma (IFNgamma)--but not IL-1beta or TNFalpha--generated nitrite in vitro. A cytokine cocktail of IL-1beta, TNFalpha, and IFNgamma synergistically enhanced nitrite production. Nitrite production was blocked by the addition of aminoguanidine (AG), suggesting that AG-inhibited cytokine mediated nitrite production. However, in an in vivo model of cholesteatoma-induced bone resorption, the osteoclast response of AG-treated mice was not statistically different from untreated controls.
  
  
  CONCLUSIONS
  All three NOS isoforms were expressed in an in vivo model of cholesteatoma-induced bone resorption with significant upregulation of NOS II throughout the study. Exogenously administered nitric oxide dose-dependently enhanced osteoclast activation in vitro. The pro-inflammatory cytokines, IL-1beta, TNFalpha, and IFNgamma, synergistically induce nitrite production, which was abrogated by treatment with the nitric oxide synthase inhibitor, AG. Although AG suppresses nitrite production in vitro, treatment had no effect on osteoclast response in vivo, suggesting that the effects of inflammatory cytokines on osteoclast response were mediated through other pathways.
authors.affiliation: Department of Otolaryngology, Washington University, St. Louis, Missouri 63110, USA.
authors.forename: Jae Y
authors.initials: JY
authors.lastname: Jung
authors.forename: Mary E
authors.initials: ME
authors.lastname: Pashia
authors.forename: Sheri Y
authors.initials: SY
authors.lastname: Nishimoto
authors.forename: Brian T
authors.initials: BT
authors.lastname: Faddis
authors.forename: Richard A
authors.initials: RA
authors.lastname: Chole
mesh_terms: D000818:Animals; D001862:Bone Resorption / Q000201:enzymology / Q000209:etiology / Q000503:physiopathology*; D002478:Cells, Cultured; D002781:Cholesteatoma / Q000150:complications / Q000473:pathology / Q000503:physiopathology*; D016207:Cytokines / Q000494:pharmacology; D004195:Disease Models, Animal; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006146:Guanidines / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D011897:Random Allocation; D014409:Tumor Necrosis Factor-alpha
chemical_list: D016207:Cytokines; D004791:Enzyme Inhibitors; D006146:Guanidines; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; C004479:pimagedine