Urokinase-plasminogen activator protects periodontal ligament fibroblast from oxidative induced-apoptosis and DNA...

Urokinase-plasminogen activator (uPA) is a serine protease expressed at high basal level in normal gingival cervical fluid. Despite its known pathologic role in tissue proteolysis in periodontitis, little is known concerning uPA physiological function in oral tissue. Recent evidence in cancer cells has implicated the uPA system in DNA repair and anti-apoptotic pathways. This study is aimed to evaluate the protective function of urokinase against oxidative DNA damage in periodontal ligament (PDL) fibroblast, and to propose a new biological role for uPA in oral cavity.

PDL cells were isolated from human wisdom teeth obtained from healthy donors. An oxidative stress model was created in which PDL cells were incubated with 20, 30, 40 and 60 μmol/L hydrogen peroxide. Twenty-four hours before and after peroxide treatment, cells were treated with uPA and amiloride. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, apoptosis by DAPI-staining and annexin V/propidium iodide assay, and DNA breaks by alkaline comet assay. For estimating DNA damage level, γ-H2AX expression was studied using flow cytometry and immunostaining.

The incubation of the peroxide-treated cells with uPA significantly increased cell viability and decreased apoptosis. A significant decrease in the number of γ-H2AX foci was seen at 30 μmol/L hydrogen peroxide in uPA-treated cells. uPA inhibition as a result of amiloride treatment, in turn, induced a reduction in cell viability. In addition, there was a significant decrease in the levels of DNA damage in uPA-treated groups as measured by the comet assay.

The present study brings support to the theory that uPA may have a protective role for periodontal tissue and could protect PDL fibroblasts from oxidative DNA damage and apoptosis.