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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02926

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Myosin light chain kinase plays a role in the regulation of epithelial cell survival

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
² Department of Molecular and Cellular Biology, SUNY Stony Brook, Stony Brook, NY 11794, USA
³ Department of Cell Biology and Anatomy, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, 1550 NW 10 Avenue M-877, Miami, FL 33136, USA

View larger version (40K): [in a new window]   Fig. 1. FACS analysis of cells treated with MLCK and ROCK inhibitors. (A) Percentage apoptosis of untransformed and Ras-transformed MCF-10A cells grown on polyHEMA or treated with inhibitors of MLCK or ROCK. Cells were plated on polyHEMA and adherent cells were treated with ML-7 (40 µM), ML-9 (40 µM) and Y-27632 (30 µM) for 16 hours. Cells were then collected, stained with annexin V FITC and propidium iodide and subjected to FACS analysis. Results presented are the mean ± s.e.m. of at least three experiments. (B) Percentage apoptosis of MDCK and MDCK Ras cells grown on polyHEMA or treated with inhibitors of MLCK or ROCK. (C) Example plot of untreated MCF-10A cells, and MCF-10A cells treated with ML-7.

View larger version (96K): [in a new window]   Fig. 2. Inhibition of MLCK by ML-7 induces activation of caspase-3 in adherent MCF-10A and MCF-10A Ras-transformed cells. Cells were grown on coverslips and treated with ML-7 (40 µM) for 16 hours. Cells were then fixed with 3% paraformaldehyde, then permeabilized and stained with Topro-3, anti caspase-3 (Cell Signaling) and anti-actin (Rhodamine-phalloidin).

View larger version (87K): [in a new window]   Fig. 3. Inhibition of MLCK by ML-9 induces activation of caspase-3 in adherent MCF-10A and MCF-10A Ras-transformed cells. Cells were grown on coverslips and treated with ML-9 (40 µM) for 16 hours. Cells were then fixed with 3% paraformaldehyde, permeabilized and stained with Topro-3, anti caspase-3 (Cell Signaling) and anti-actin (Rhodamine-phalloidin).

View larger version (96K): [in a new window]   Fig. 4. Inhibition of ROCK by Y-27632 does not activate caspase-3 in adherent MCF-10A and MCF-10A Ras-transformed cells. Cells were grown on coverslips and treated with Y27632 (30 µM), for 16 hours. Cells were then fixed with 3% paraformaldehyde, permeabilized and stained with Topro-3, anti caspase-3 (Cell Signaling) and anti-actin (Rhodamine-phalloidin).

View larger version (75K): [in a new window]   Fig. 5. Expression of kinase-dead MLCK activates caspase-3 in MCF-10A cells. (A) Normal and Ras-transformed MCF10A cells transfected with a kinase-dead MLCK undergo apoptosis. Cells were grown on coverslips, transfected with a FLAG-tagged kinase-dead non-muscle (NM MLCK KD) or kinase-dead smooth muscle MLCK (SM MLCK KD) using Lipofectamine 2000 (Invitrogen). Cells were grown for 24 hours, fixed in 3% paraformaldehyde and stained with anti-FLAG polyclonal antibody (Sigma), Topro-3 and anti-actin (Rhodamine-phalloidin).

View larger version (37K): [in a new window]   Fig. 6. Overexpression of dominant-negative FADD can inhibit apoptosis in MCF-10A cells treated with MLCK or actin polymerization inhibitors. FACS analysis of MCF-10A (A) and MCF-10A Ras-transformed (B) cells expressing pBabe DN FADD after plating on polyHEMA or treatment with ML-7, ML-9 or Y-27632 for 16 hours. Results are the mean ± s.e.m. of at least three experiments.

View larger version (62K): [in a new window]   Fig. 7. Decreased activation of ß1 integrin in MCF10A cells after inhibition of myosin II, actin polymerization and MLCK. Untransformed MCF-10A (A) or Ras-transformed MCF-10A cells (B) were treated with MLCK, myosin II and actin polymerization inhibitors, as indicated. Cells were grown on coverslips and treated for 2 hours, then fixed in 3% paraformaldehyde and stained with an anti-ß1-integrin antibody (clone HUTS-21), which recognizes integrins in their active state, Topro-3 and anti-actin (Rhodamine-phalloidin). (C) Western blot showing level of ß1 integrin after 2 hours of treatment with inhibitors.

View larger version (45K): [in a new window]   Fig. 8. Addition of a ß1-integrin-activating antibody, 9EG7, rescues cells from apoptosis as a result of MLCK or actin polymerization inhibition. MCF-10A (A) and MCF-10A Ras-transformed (B) cells were plated with 20 µg/ml 9EG7 for 24 hours and subsequently treated with ML-7, ML-9 and LA. (C) Phospho-FAK is decreased in cells treated with inhibitors, and restored in MCF-10A and MCF-10A Ras cells pre-treated with 9EG7, then incubated with Blebbistatin (BB), LA or ML-7 for 1 hour. Total FAK levels remain the same in each cell line with treatments (C).

View larger version (15K): [in a new window]   Fig. 9. Model for adhesion-dependent and adhesion-independent integrin signaling. In model 1, survival signals are generated by both adhesion and growth factors, as seen in untransformed cells. Model 2 illustrates a possible mechanism of eliminating the requirement for adhesion by Ras-transformed cells, but signaling events that are still dependent on myosin II. We propose that Ras-transformed cells can activate myosin II through MLCK, leading to integrin activation and associated survival signals.