2-hydroxyethyl methacrylate (HEMA), deriving from polymerized resinous biomaterials, can diffuse at gingival and tooth pilp level. Our study aimed to investigate the human gingival fibroblasts (HGFs) response to a low HEMA concentration, in terms of inflammatory genes expression and signal transduction proteins activation. Cultured HGFs were exposed to 3 mM HEMA for 0, 24 or 96h. At each experimental point Reactive Oxygen Species (ROS) production were investigated by flow cytometry; Tumor Necrosis Factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) gene expression were determined by RT-PCR and prostaglandin E2 (PGE2) production was detected by an enzyme immunoassay. Since HEMA treatment decreased vitality and induced apoptosis in HGFs, at the same experimental points PKCs activation, Bax and Apaf-cyt C immunoprecipitate expression were evaluated by Western Blotting analysis and NOS activity by a specific "in vitro" assay. 24h HEMA incubation induces an increase in ROS production persisting up to 96 h. 24 and 96h HEMA treatment significantly enhances TNF-alpha and COX-2 gene expression compared to control in a time-dependent manner. 96h HEMA incubation significantly increases PGE2 concentration in the culture medium, PKC alpha expression and sctivation, iNOS activity and Bax expression when compared to control. Interestingly, a reduced percentage of apoptotic cells and a reduced ROS production is evidenced in the presence of bisindolylmaleide VIII, a PKC alpha pharmacological inhibitor, giving specificity to our data. All in all, these results suggest that 24 or 96h 3 mM HEMA treatment induces in HGFs an inflammatory response modulated by ROS production, PKC alpha activation and TNF-alpha gene expression increase. Such pathways converge on the up-regulation of COX-2 gene expression, which leads to the increase of PGE2 release, inducing cell proliferation arrest and apoptosis occurrence.
PKC alpha mediated specific gene expression occurs in the inflammatory and stress response of human gingival fibroblasts to 2-hydroxyethyl mathacrylate
Rapino M;
2012
Abstract
2-hydroxyethyl methacrylate (HEMA), deriving from polymerized resinous biomaterials, can diffuse at gingival and tooth pilp level. Our study aimed to investigate the human gingival fibroblasts (HGFs) response to a low HEMA concentration, in terms of inflammatory genes expression and signal transduction proteins activation. Cultured HGFs were exposed to 3 mM HEMA for 0, 24 or 96h. At each experimental point Reactive Oxygen Species (ROS) production were investigated by flow cytometry; Tumor Necrosis Factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) gene expression were determined by RT-PCR and prostaglandin E2 (PGE2) production was detected by an enzyme immunoassay. Since HEMA treatment decreased vitality and induced apoptosis in HGFs, at the same experimental points PKCs activation, Bax and Apaf-cyt C immunoprecipitate expression were evaluated by Western Blotting analysis and NOS activity by a specific "in vitro" assay. 24h HEMA incubation induces an increase in ROS production persisting up to 96 h. 24 and 96h HEMA treatment significantly enhances TNF-alpha and COX-2 gene expression compared to control in a time-dependent manner. 96h HEMA incubation significantly increases PGE2 concentration in the culture medium, PKC alpha expression and sctivation, iNOS activity and Bax expression when compared to control. Interestingly, a reduced percentage of apoptotic cells and a reduced ROS production is evidenced in the presence of bisindolylmaleide VIII, a PKC alpha pharmacological inhibitor, giving specificity to our data. All in all, these results suggest that 24 or 96h 3 mM HEMA treatment induces in HGFs an inflammatory response modulated by ROS production, PKC alpha activation and TNF-alpha gene expression increase. Such pathways converge on the up-regulation of COX-2 gene expression, which leads to the increase of PGE2 release, inducing cell proliferation arrest and apoptosis occurrence.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
