Nitric oxide production by a human osteoblast cell line stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide.
OBJECTIVE
Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).


METHODS
Cells were stimulated directly with A. actinomycetemcomitans lipopolysaccharide or pretreated with the following l-NIL (an iNOS inhibitor), anti-CD14, Toll-like receptor 2 (TLR2), or TLR4 antibody before stimulation with A. actinomycetemcomitans lipopolysaccharide. The role of the cyclic nucleotides was assessed by pretreating the cells with the following; ODQ (a guanylyl cyclase inhibitor); SQ22536 (an adenylyl cyclase inhibitor); db-cAMP (a cyclic adenosine monophosphate analog); br-cGMP (a cyclic guanosine monophosphate analog); forskolin (an adenylyl cyclase activator), IBMX [a non-specific phosphodiesterase (PDE) inhibitor], or KT5720 [a protein kinase A (PKA) inhibitor]. The cells were also preincubated with genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], BPB [a phospholipase A2 (PLA2) inhibitor], and NDGA (a lipoxygenase inhibitor). The iNOS activity and nitrite production in the cell cultures were determined spectrophotometrically.


RESULTS
The results showed that A. actinomycetemcomitans lipopolysaccharide stimulated both iNOS activity and nitrite production by HOS cells; this was reduced by l-NIL, anti-CD14, or anti-TLR4 antibody, SQ22536, KT5720, genistein, bisindolylmaleimde, BPB, and NDGA, but was enhanced by db-cAMP, IBMX, and forskolin.


CONCLUSIONS
These results therefore suggest that A. actinomycetemcomitans lipopolysaccharide may induce the production of NO by HOS cells via a CD14-TLR4 molecule complex, a cAMP-PKA pathway, as well as by a PTK, PKC, PLA2, and lipoxygenase-dependent mechanism.
Sosroseno
W
W
School of Dentistry, AIMST University, Semeling, Bedong, Kedah Darul Aman, Malaysia. wsosroseno@yahoo.com
Bird
P S
PS
Seymour
G J
GJ
D016976:Aggregatibacter actinomycetemcomitans / Q000276:immunology*; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D008084:Lipoxygenase / Q000378:metabolism; D016859:Lipoxygenase Inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism; D010740:Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000276:immunology
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016859:Lipoxygenase Inhibitors; D010726:Phosphodiesterase Inhibitors; D047428:Protein Kinase Inhibitors; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D000242:Cyclic AMP; D008084:Lipoxygenase; D052247:Nitric Oxide Synthase Type II; D011494:Protein Kinases; D010740:Phospholipases; D006152:Cyclic GMP