A possible role for nitric oxide in osteoclastogenesis associated with cholesteatoma

Jae Y. Jung, Mary E. Pashia, Sheri Y. Nishimoto, Brian T. Faddis, Richard A. Chole

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13 Scopus citations


Hypothesis: 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 γ (IFNγ)-but not IL-1β or TNFα-generated nitrite in vitro. A cytokine cocktail of IL-1β, TNFα, and IFNγ 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-1β, TNFα, and IFNγ, 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.

Original languageEnglish
Pages (from-to)661-668
Number of pages8
JournalOtology and Neurotology
Issue number5
StatePublished - Sep 2004


  • Cholesteatoma
  • Inflammation
  • Nitric oxide
  • Osteoclast
  • Osteolysis


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