Type I collagen (COL I) is the predominant collagen in the extracellular matrix of periodontal ligament (PDL), and its expression in PDL fibroblasts (PLF) is sensitive to mechanical force. However, the mechanism by which PLF induces COL I to respond to mechanical force is unclear. This study examined the nature of human PLF in mediating COL I expression in response to centrifugal force. Signal transduction pathways in the early stages of mechanotransduction involved in the force-driven regulation of COL I expression were also investigated. Centrifugal force up-regulated COL I without cytotoxicity and activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. ERK and JNK inhibitor blocked the expression of COL I but p38 kinase inhibitor had no effect. Centrifugal force activated activator protein-1 (AP-1) through dimerization between c-Fos and c-Jun transcription factors. ERK and JNK inhibitors also inhibited AP-1-DNA binding, c-Fos nuclear translocation, and c-Jun phosphorylation that were increased in the force-exposed PLF. Further, transfecting the cells with c-Jun antisense oligonucleotides almost completely abolished the force-induced increase of c-Jun phosphorylation and COL I induction. Our findings suggest that mechanical signals are transmitted into the nucleus by ERK/JNK signaling pathways and then stimulate COL I expression through AP-1 activation in force-exposed human PLF.
It has been proposed that continuously generated hydrogen peroxide (H(2)O(2)) inhibits typical apoptosis and instead initiates an alternate, apoptosis-inducing factor (AIF)-dependent process. Aside from the role of AIF, however, the detailed morphological characterization of H(2)O(2)-induced cell death is not complete. This study examined the cellular mechanism(s) by which the continuous presence of H(2)O(2) induces cell death. We also further analyzed the precise role of AIF by inhibiting its expression with siRNA. Exposure of cells to H(2)O(2) generated by glucose oxidase caused mitochondrion-mediated, caspase-independent cell death. In addition, H(2)O(2) exposure resulted in cell shrinkage and chromatin condensation without nuclear fragmentation, indicating that H(2)O(2) stimulates a pyknotic cell death. Further analysis of AIF-transfected cells clearly demonstrated that nuclear translocation of AIF is the most important event required for nuclear condensation, phosphatidyl serine translocation, and ultimately cell death in H(2)O(2)-exposed cells. Furthermore, ATP was rapidly and severely depleted in cells exposed to H(2)O(2) generated by glucose oxidase but not by H(2)O(2) added as a bolus. Suppression of the H(2)O(2)-mediated ATP depletion by 3-aminobenzamide led to a significant increase of nuclear fragmentation in glucose oxidase-exposed cells. Collectively, these findings suggest that an acute energy reduction by H(2)O(2) causes caspase-independent and AIF-dependent cell death.
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