Disease-associated fibronectin fragment generates nitric oxide in human periodontal cells

Objectives: Inflammation and bacterial infection of oral tissues results in the degradation of the extracellular matrix (ECM) leading to the release of fibronectin fragments. One such fibronectin fragment (FnF) which is defective in binding to heparin, has previously been shown to cause apoptosis of human periodontal ligament (PDL) cells. Often the degraded products of the ECM induce cells to produce nitric oxide (NO) which is important for both pro- and anti-apoptotic processes. The purpose of this study was to determine whether FnF can induce nitric oxide synthase (NOS) in human PDL cells to produce NO.

Methods: NO production was assayed using Griess reagent and induction of inducible NOS (I-NOS) and levels of the stress protein JNK-1 were confirmed by immunoblotting and immunofluorescence.

Results: Cells treated with FnF showed strong immunofluorescence for I-NOS and exhibited a dose- and time- dependent NO production. Pretreatment of cells with inhibitors for I-NOS before FnF treatment greatly reduced NO production. Also, FnF significantly increased NO production from these cells when coincubated with LPS from Porphyromonas gingivalis. Coincubation of these cells with interferon gamma, a known inducer of I-NOS, and the Porphyromonas LPS greatly enhanced NO production in presence of FnF. Furthermore, cells transfected with an I-NOS cDNA showed increased NO levels, whereas transfection with I-NOS siRNA substantially reduced this production upon FnF treatment. To determine whether JNK-1 is important for induction of I-NOS by FnF, we either pretreated cells with SB203580, a known inhibitor of JNK, or transfected cells with JNK-1 siRNA. These treatments showed that NO generation was suppressed in either case. However, overexpression of JNK-1 increased the production of NO upon FnF treament. Also, FnF treatment increased the phosphorylation of JNK-1.

Conclusion: FnFs associated with inflammation and bacterial infection of oral tissues induce cells to generate NO in a JNK-1 dependent manner.

NIHRO1DE013725-to-YLK