Decrease in saliva causes serious problems in clinical dentistry. There have been previous reports on tissue injuries of parotid glands inducing a dedifferentiation signal that causes the loss of function of acinar cells. Since both inhibitors for Src family kinase (SFK) and p38 MAP kinase (p38 MAPK) suppressed the dedifferentiation, SFK-p38 MAPK pathway was considered essential for the signaling. Even though Src and Yes were expressed in the parotid glands, the increase in phosphorylation level was not detected for conserved tyrosine residue in the kinase domain of Src or Yes that promotes kinase activity, following tissue injuries. In this study, the kinase activities were assayed by using the specific substrate for SFK to examine whether the enhancement of kinase activity of SFK was eventuated by tissue injuries. As a result, the activity of the total SFK was elevated after the cellular damage and was sustained for 2 h. To determine the activated SFK member, the SFK activity was examined by immunoprecipitation method with specific antibodies against each SFK member. Elevation of kinase activities of both Src and Yes was detected after cell damages. The elevation ratio of Src activity was higher than Yes, suggesting that the contribution of Src in the dedifferentiation signaling is higher.Diphenyleneiodonium, an NADPH oxidase inhibitor, suppressed the activation of SFK and p38 MAPK, and the expression of claudin-4, a dedifferentiation marker. These results suggest that the activation of SFK is induced by oxidative stresses and is essential for p38 MAPK-mediated dedifferentiation signaling.
Ceramide is generated by the hydrolysis of membrane sphingomyelin by sphingomyelinase and is implicated in multiple signaling pathways, including those regulating differentiation, inflammation and immune responses. Excess formation of prostaglandin E 2 (PGE 2 ) is thought to increase susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases. We investigated the inhibitory effect of C 2 -ceramide, a short-chain ceramide analog, on the PGE 2 -stimulated accumulation of cAMP in human gingival fibroblasts. In human gingival fibroblasts pre-treated with C 2 -ceramide for 18 h, the PGE 2 -stimulated accumulation of cAMP was reduced, but an inactive C 2 -ceramide analog had no such effect. The accumulation of cAMP induced by EP2 and EP4 receptor agonists (ONO-AE1-259 and ONO-AE1-329, respectively) was inhibited in cells treated with C 2 -ceramide. However, treatment with C 2 -ceramide had no effect on the expression of mRNAs encoding the EP2 and EP4 receptors. Accumulation of cAMP could be induced by cAMP-elevating agents (forskolin, isobutylmethylxanthine and mastparan) but was not reduced by treatment with C 2 -ceramide. These observations suggest that C 2 -ceramide attenuates PGE 2 receptor function and consequently inhibits the accumulation of cAMP in human gingival fibroblasts.Prostaglandins are lipid mediators that are involved in many physiological and pathophysiological processes. The excess formation of prostaglandin E 2 (PGE 2 ) has been described in a number of conditions characterized by increased susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases (8,17,25,29). PGE 2 mediates its biological functions via binding to four types of membrane-bound, G protein-coupled receptors, termed E prostanoid (EP)1 to EP4 (6, 22). EP receptors stimulated by ligand binding activate different signal transduction pathways. Activation of the EP1 receptor raises intracellular Ca 2+ levels. Activation of EP2 and EP4 receptors increases intracellular cAMP levels by activating adenylate cyclase via G s proteins. Activation of the EP3 receptor reduces or increases cAMP levels by activating inhibitory G (G i ) or stimulatory G (G s ) proteins depending on the particular splice variant expressed by the cell (15). In human gingival fibroblasts, PGE 2 inhibited the DNA synthesis and proliferation (1, 37), and downregulated intercellular adhesion molecule-1 expression by cAMP-dependent signaling pathways (24). Therefore, cAMP is thought to be the main intracellular second messenger for response to PGE 2 , and is the crucial modulator of the functional activity of inflammation. Ceramide, an intracellular second messenger in the sphingomyelin transmembrane signaling pathway, can be generated by the cleavage of sphingomyelin by sphingomyelinase or by de novo biosynthesis by ceramide synthase (14). Increases in intracellular ceramide have been reported in many
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