Abstract
Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.
Highlights
Function such as differentiation, proliferation, and migration (1)
Application of high amplitude tensile forces through collagen-coated magnetite beads in fibroblasts, which is a good model for the forces that are delivered to connective tissue cells in vivo (7), induces p38 and eIF2␣ phosphorylation (9) and cell death (10). eIF2␣ is regulated in the unfolded protein response (UPR) as cells progress from physiological protein translation to compensatory responses to exogenous forces
Mechanical Force Induces Apoptosis—Robust time-dependent increases in the active form of caspase 3 were observed in mouse embryonic fibroblasts (MEFs) and in Human gingival fibroblasts (HGFs) exposed to tensile force (Fig. 1a)
Summary
Reagents—JC-1 and mag-fura-2/AM were purchased from Molecular Probes (Eugene, OR). Magnetite beads, bovine serum albumin (BSA), poly-L-lysine, fibronectin, thapsigargin, tunicamycin, staurosporine, carbonyl cyanide 3-chlorophenylhydrazone, and camptothecin were obtained from Sigma. After force induction, cells were washed with PBS, and lysates were collected in Laemmli sample buffer for determination of protein concentration and analysis. After force application, paraformaldehyde-fixed cells were permeabilized with Citrate buffer (0.1% Triton X-100 in 0.1% sodium citrate) for 15 min at room temperature, washed twice with PBS, and stained with the TUNEL reagent mixture for at least 1 h at 37 °C. They were washed with PBS and counterstained with 4Ј,6-diamidino-2-phenylindole. All experiments were performed at least three times in triplicate
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