Lipid A‐associated proteins from Porphyromonas gingivalis stimulate release of nitric oxide by inducing expression of inducible nitric oxide synthase

Abstract

The purpose of this study was to examine the effects of lipid A-associated proteins from Porphyromonas gingivalis, a major cause of inflammatory periodontal disease, on the production of nitric oxide and expression of inducible nitric oxide synthase in the murine macrophage cell line, RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. gingivalis lipid A-associated protein-induced nitric oxide production. The lipid A-associated proteins from P. gingivalis 381 were prepared by standard hot phenol-water extraction of endotoxin isolated by the butanol method. Nitric oxide production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of inducible nitric oxide synthase and analysis of reverse transcription-polymerase chain reaction products were carried out. We found that P. gingivalis lipid A-associated proteins can induce inducible nitric oxide synthase expression and stimulate the release of nitric oxide without additional stimuli, and we demonstrated that multiple signaling pathways, such as nuclear factor-kappaB, microtubule polymerization, protein tyrosine kinase, protein kinase C, and mitogen-activated protein kinase cascades, are involved in P. gingivalis lipid A-associated protein-stimulated nitric oxide production. The production of nitric oxide required l-arginine. The present study clearly shows that P. gingivalis lipid A-associated proteins fully induced inducible nitric oxide synthase expression and nitric oxide production in RAW264.7 cells in the absence of other stimuli. The ability of P. gingivalis lipid A-associated proteins to promote the production of nitric oxide may be important in the pathogenesis of inflammatory periodontal disease.