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First published online 15 July 2003
doi: 10.1242/jcs.00660

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Caspase activation during phorbol ester-induced apoptosis requires ROCK-dependent myosin-mediated contraction

Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1 section 1 Jen-Ai Road, Taipei 100, Taiwan, Republic of China

View larger version (38K): [in a new window]   Fig. 1. PMA-induced pro-apoptotic signals are associated with early detection of membrane blebbing and MLC phosphorylation, followed by caspase-3 activation. (A) TF-1 cells (1x106) were treated with PMA (32 nM) in 10% (v/v) heat-inactivated FBS-containing RPMI medium in the presence of GM-CSF. Cell lysates obtained from cells that remained in suspension were analyzed for caspase-3 activity. Results are expressed relative to those obtained in the untreated control, and are the mean±s.d. of two independent experiments. (B) TF-1 cells were pre-incubated with cycloheximide (CHX, 50 µg/ml), or staurosporine (STS, 200 nM) as indicated for 30 minutes prior to PMA treatment. Viability was determined by Trypan Blue exclusion method and was expressed relative to untreated cells. Results are the mean±s.d. (n=3). (C) TF-1 cells treated with PMA for the indicated time points were imaged by phase-contrast microscopy and harvested for immunoblot analysis. (D) Whole cell lysates (50 µg) were resolved by SDS-PAGE and blotted with antibodies specific for phosphoERK1/2, phosphoMLC, MLC, caspase-3 and ROCKI.

View larger version (32K): [in a new window]   Fig. 2. Serum or LPA in medium affects ROCK-mediated MLC phosphorylation, which is closely related to PMA-induced apoptosis. PMA, specific ROCK inhibitor (Y27632; 20 µM), 10% (v/v) heat-inactivated FBS or LPA (10 µM) was added to the medium separately or in different combinations as indicated. (A) After 12 hours, viability was determined using the Trypan Blue exclusion method. Results are the means±s.d. from three independent experiments. (B) In parallel, cells treated with PMA for 8 hours were harvested for immunoblotting with antibodies specific for phosphoERK1/2, phosphoMLC and MLC.

View larger version (47K): [in a new window]   Fig. 3. ROCK activity is required for PMA-induced MLC phosphorylation and membrane contraction, which precedes caspase activation. (A) TF-1 cells were either left untreated or pre-incubated with the indicated blocker for 30 minutes: pan-caspase inhibitor (Boc-D-FMK; 50 µM), specific ROCK inhibitor (Y27632; 20 µM, for 1 hour) or F-actin polymerization inhibitor (Latrunculin B; 0.5 µM). Cells were then exposed to PMA for 2 hours and imaged by phase-contrast microscope for photography. The cell viability in each set of cultures was determined by Trypan Blue staining after exposure to PMA for 12 hours. (B) TF-1 cells were pre-treated with Y27632 (20 µM) or ML-7 as indicated, followed by addition of PMA to the medium. Cells were harvested 8 hours later and resolved by SDS-PAGE, followed by immunoblotting with antibodies specific for phosphoMLC, MLC and phosphoERK1/2. After 12 hours exposure to PMA, viability was determined using the Trypan Blue exclusion method. (C) The effects of Y27632 (20 µM) or ML-7 (20 µM) on PMA-induced apoptosis were assessed by DNA laddering. NT, not treated.

View larger version (11K): [in a new window]   Fig. 4. PMA-induced activation of caspase-3 is dependent on ROCK activation, F-actin polymerization and myosin ATPase activity. TF-1 cells were treated with PMA in the absence or presence of different pharmacological inhibitors as indicated (ROCK inhibitor: Y27632, 20 µM; F-actin polymerization inhibitor: Latrunculin B (L.B), 0.5 µM; myosin ATPase inhibitor: BDM, 5 µM). After 8 hours, cells were harvested and analyzed for caspase-3 activity as described in the Materials and Methods. Results are expressed as the increase in 405 nm absorbance by subtracting the reading values from those obtained in cells without PMA treatment from four independent experiments.

View larger version (10K): [in a new window]   Fig. 5. Inhibition of ROCK abrogates the requirement of adhesion for survival. TF-1 cells were pre-incubated without or with Y27632 in serum-free medium for 1 hour. Cells were then plated on regular or hydrogel-coated plates following PMA treatment for 12 hours. Cell viability was also performed as described earlier.

View larger version (11K): [in a new window]   Fig. 6. Effect of expression of dominant-active forms of RhoA and ROCK on PMA-induced apoptosis in serum-free condition. All cells were transiently transfected with 1.5 µg of GFP expression vector and 4.5 µg of the indicated plasmid or control vector. After transfection for 48 hours, cells were washed twice with PBS, and then suspended in serum-free medium containing GM-CSF without or with PMA (32 µM) for 8 hours. GFP-positive cell numbers in suspension were determined and are expressed as relative to the cells transfected with the control vector. All results are the mean±s.d. (n=4).

View larger version (36K): [in a new window]   Fig. 7. Activation of caspase-8 and -10 in PMA-induced apoptosis. (A) TF-1 cells were treated with PMA for different time intervals (0-12 hours). Whole cell lysates (50 µg) were resolved by SDS-PAGE followed by immunoblotting with antibodies specific for caspase-8 and caspase-10. (B) TF-1 cells were pre-incubated with different caspase inhibitors (5 µM) for 30 minutes as indicated prior to PMA treatment. The corresponding inhibitor of each caspase is z-DEVD-fmk for caspase-3, z-IETD-fmk for caspase-8, z-LEHD-fmk for caspase-9 and z-AEVD-fmk for caspase-10. After 8 hours, cells were analyzed for caspase-3 activity as described in the legend to Fig. 4. (C) Following pretreatment with z-DEVD-fmk (10 or 50 µM) for 30 minutes, TF-1 cells were treated with PMA for 9 hours and harvested for immunoblot analysis using antibodies specific for ROCKI, caspase-8 and -10.

View larger version (89K): [in a new window]   Fig. 8. ROCK-mediated signal is required for processing of procaspases-8 and -10 in PMA-treated TF-1 cells. TF-1 cells were either left untreated or pre-incubated with specific ROCK inhibitor (Y27632; 20 µM), Latrunculin B (L.B, 0.5 µM) or MLCK inhibitor (ML-9; 20 µM), as indicated. Eight hours after PMA treatment, cells were harvested and analyzed by immunoblotting with antibodies specific for caspase-3, -8 and -10. NT, not treated.

View larger version (41K): [in a new window]   Fig. 9. Involvement of FADD in PMA-induced apoptosis. (A) D2 or TF-1 cells transfected with procaspase-10 C/S-flag were either untreated or treated with PMA for 3 hours (D2) or 5 hours (TF-1) as indicated. For DISC analysis, the post-nuclear supernatant from transfected cells was immunoprecipitated with anti-flag M2 beads or with normal mouse IgG followed by addition of protein A-Sepharose beads. The resulting protein complexes were separated by SDS-PAGE, and analyzed by immunoblotting with antibodies for flag and FADD. (B) D2 cells transfected with procaspase-10 C/S-flag were treated with PMA for 30 minutes, the pro-apoptotic suspension cells were then transferred to a new dish without or with Y27632 for another 2.5 hours. DISC analysis was then performed as described above. The lysates without coimmunoprecipitation were directly subjected to the same immunoblotting analysis and indicated as 'Input'. Abbreviations: NT, non-treated; S, cells in suspension representing PMA-induced apoptotic cells; A, attached cells representing PMA-induced pro-differentiation or survived cells; IgG H, heavy chain; IgG L, light chain; IP, immunoprecipitation.

View larger version (23K): [in a new window]   Fig. 10. Model for PMA-induced apoptosis involving the signal from the RhoA/ROCK pathway. During PMA treatment, a concurrent signal from LPA or serum upregulates the RhoA/ROCK/MLC phosphorylation pathway, which cooperates with the PMA/PKC signal to generate a membrane contraction force that leads to activation of caspase-8 and -10. In this model, we propose that additional pathways including blocking myosin phosphatase by PKC-mediated upregulation of CPI, the negative control on MLC phosphorylation by cell adhesion, and the subsequent death-receptor-dependent or -independent pathway for activation of caspases-8 and -10, interplay with RhoA/ROCK pathway in PMA-induced apoptosis. Dashed lines represent the pathways that remain to be verified.

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