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First published online July 23, 2007
doi: 10.1242/10.1242/jcs.003566

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PKC and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin 5 but not its tailless mutant

¹ Cancer Research Institute, College of Medicine, Seoul National University, 28, Yeongeon-dong, Jongno-gu, Seoul 110-799, Korea
² Department of Tumor Biology, Seoul National University, 28, Yeongeon-dong, Jongno-gu, Seoul 110-799, Korea
³ Department of Molecular and Clinical Oncology, Seoul National University, 28, Yeongeon-dong, Jongno-gu, Seoul 110-799, Korea
⁴ Laboratory of Angiogenesis and Chemoprevention, CPMDRC, College of Oriental Medicine, Kyunghee University, 1 Hoegidong, Dongdaemugu, Seoul 131-701, Korea

View larger version (89K): [in this window] [in a new window]   Fig. 1. Cell-cell contact loss of RIE1-5, but not RIE1-5/1, cells after OXO treatment. (A) Stable cell lines ectopically expressing integrin 5 (RIE1-5) or tailless 5 (RIE1-5/1) were maintained, as described in Materials and Methods. Images for subconfluent cells in normal culture media were taken using a microscope equipped with a digital camera. Both cell lines generally form colonies. (B) Whole cell lysates from subconfluent cells were prepared for immunoblotting using an antibody against an extracellular region (5exo) or the cytoplasmic tail (5cyto) of the human integrin 5 subunit, or -tubulin. Data shown represent at least three independent experiments. (C) Chemical structure of 6-(1-oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone (OXO). (D,E) Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. Once confluent monolayers had been formed by incubation in 5% CO2 at 37°C, cells were treated with either vehicle (DMSO) or OXO (10 µM) for 24 hours, prior to being immunostained for ZO1 (D) or -catenin (E). Data shown are representative of three different experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Signal activity specifically impaired in RIE1-5/1 cells by OXO treatment. (A,B) Subconfluent (7080% confluent) cells in 60 mm culture dishes were treated with either DMSO or 10 µM OXO for 24 hours. Protein amounts in whole cell lysates were normalized before standard western blotting for the indicated molecules. Data shown are representative of three independent experiments.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Inhibition of PI3K and SFK blocked OXO-mediated scattering of RIE1-5 cells, but did not affect cell-cell contacts between RIE1-5/1 cells. Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. Once cells formed monolayers, they were not pretreated or pretreated with LY294002 (LY, 20 µM), PP2 (10 µM), or PP3 (10 µM), 30 minutes before DMSO or 10 µM OXO treatment. Cells were processed for immunostaining against ZO1, 24 hours later, as described in Materials and Methods. Data shown are representative of three independent experiments.

View larger version (87K): [in this window] [in a new window]   Fig. 4. Differential cofilin phosphorylation and peripheral actin reorganization in RIE1-5 and RIE1-5/1 cells. (A) Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. After cells had adhered and spread typically, they were treated with either DMSO or 30 µM OXO for 30 minutes. Cells were then processed for actin staining using phalloidin-conjugated with Rhodamine, as described in Materials and Methods. Note that 30 µM OXO treatment disrupted actin filament organization only in RIE1-5 cells. (B) Cells on normal culture medium-coated coverslips were incubated with 20 µM DCHF-PBS for 30 minutes, prior to washing and visualization of ROS-positive cells by fluorescent microscopy. (C,D) Cells (at 7080% confluence) in 60 mm culture dishes were not pretreated or pretreated with the indicated pharmacological inhibitors, as in Fig. 3, prior to treatment with DMSO or 10 µM OXO for 24 hours (C) or with DMSO (i.e. 0 µM of cytochalasin D) or cytochalasin D at the indicated concentrations for 1 hour (D). Whole cell lysates were then prepared and normalized protein amounts were used in standard western blotting for the indicated molecules. Note that RIE1-5/1 cells showed more sustained pS3cofilin levels than RIE1-5 cells did, even after treatment with cytochalasin D. (E) Cells on normal culture medium-coated coverslips were treated with DMSO or cytochalasin D (0.05 or 0.2 µM) for 1 hour, prior to actin staining as in A. (F) Cells were manipulated as described in the legend of Fig. 3, with the exception of staining for actin using phalloidin-conjugated with Rhodamine. Data shown are representative of three independent experiments.

View larger version (81K): [in this window] [in a new window]   Fig. 5. OXO-mediated cell-cell contact loss of RIE1-5 cells involves pS643PKC-dependent dynamic peripheral actin reorganization. (A,B) RIE1-5 cells (at 7080% confluence) on 10% FBS-DMEM-H-coated coverslips were treated with DMSO or 10 µM rottlerin for 24 hours (A), or with DMSO, 10 µM OXO alone or OXO plus rottlerin (10 µM OXO + 10 µM rottlerin) for 24 hours (B). Cells were then stained for actin (A) or immunostained for -catenin (B), as explained in Materials and Methods. (C-E) Cells were infected with adenovirus for GFP (Ad-cont), dominant negative K376A PKC (Ad-DN PKC) or K368R PKC (Ad-DN PKC), or wild-type PKC (Ad-WT PKC) or PKC (Ad-WT PKC) for 8 hours. After infection, media were replaced with fresh culture media. DMSO or 10 µM OXO was then treated for 24 hours, prior to immunofluorescent staining against ZO1 (left panels) or -catenin (right panels; C,D), or stained for actin using phalloidin-conjugated Rhodamine (E). (F) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Con), K376A DN PKC (DN), or WT PKC (WT), for 8 hours. After the viruses had been washed out, cells were treated with DMSO or 10 µM OXO for 24 hours, before preparation of whole cell lysates. An equal amount of proteins was subjected to standard western blotting for the indicated molecules. (G) RIE1-4 cells were manipulated as in (C) prior to ZO1 immunofluorescent staining. Data shown are representative of three independent experiments.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Dynamic reorganization of peripheral actin filament and cell-cell contact loss of RIE1-5 cells depend on PKC-dependent cofilin dephosphorylation after OXO treatment. (A-C) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Cont), K376A DN PKC (DN), or WT PKC (WT), for 8 hours (A). Cells were infected or transiently transfected with adenovirus for K368R DN or WT PKC (B) or K437R or WT PKC-HA (C), respectively. (D-I) Cells on 10% FBS-DMEM-H-coated glass coverslips (D,E) or in 60 mm culture dishes (F,G) were infected with retrovirus for control empty vector (Rt-cont), inactive hSSH1L-CS mutant (Rt-hSSH1L-CS mutant), or wild-type hSSH1L (Rt-hSSH1L WT) for 24 hours. RIE1-5 cells were transiently transfected with control (Mock), (HA)3-Akt WT (H), LIMK1, or ROCK1 (I) plasmids, 24 hours before OXO treatment. After 24 hours without or with OXO treatment, cell lysates were prepared. An equal amount of proteins was used in standard western blotting for the indicated molecules (A,B,C,F,G,H,I). Cells on coverslips were stained for actin (D,E) or immunostained for cofilin or pS3cofilin (J). Data shown are representative of three independent experiments.

View larger version (52K): [in this window] [in a new window]   Fig. 7. PKC mediates signaling from integrin 5 to focal adhesion molecules. (A) Cells were trypsinized, collected, suspended into DMEM-H-1% BSA, and rotated (60 rpm) at 37°C for 45 minutes. Then half of cells was kept in suspension and the other half was replated onto fibronectin-coated (10 µg/ml) 60 mm dishes for 2 hours. After incubation, whole cell lysates were prepared for immunoblots for the indicated molecules. (B) Whole cell extracts from the cells without or with OXO treatment were prepared and immunoprecipitated with anti-integrin 5 (clone P1D6). The immunoprecipitates and lysates were immunoblotted for pS643PKC or PKC. (C) Whole cell extracts from RIE1-5 cells were immunoprecipitated with anti-PKC. The PKC immunoprecipitates were incubated in a reaction buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 50 µM ATP and 1 mM DTT) without or with the PKC-depleted extracts (Extracts*; 10 µg protein/reaction) in the absence or presence of 10 µM OXO for 30 minutes at 25°C with shaking. After incubation, SDS-PAGE sample buffer was added to stop the reaction before immunoblotting for pS643PKC and PKC (upper panels). For in vitro PKC kinase assay (lower panel), determination of phosphorylation of Ser/Thr in the substrate by recombinant PKC (recPKC) using HTScanTM PKC kinase assay kit was performed following the manufacturer's protocols. The primary antibody of the kit and proper secondary antibody were used for immunoblots. (D) RIE1-5 cells were infected with adenovirus (Ad-Virus) encoding for a control protein (i.e. GFP, Cont) or WT PKC for 12 hours. Twenty four hours after the infection, cells were maintained in suspension (Sus) or replated onto fibronectin-coated (10 µg/ml; Fn) 60 mm dishes for 2 hours prior to cell lysates preparation and standard western blotting for the indicated molecules. (E,F) Whole cell lysates prepared as in Fig. 4C,D were normalized and used for standard western blotting for the indicated molecules. (G) RIE-4 or parental RIE1 WT cells in normal culture were harvested for integrin 4 and -tubulin immunoblots (upper panel). RIE1-4 cells were infected with control (Ad-cont) or DN PKC (Ad-DN PKC) adenovirus and treated with OXO (10 µM for 24 hours) prior to harvesting and immunoblotting for the indicated molecules. Data shown are representative of at least three independent experiments.

View larger version (137K): [in this window] [in a new window]   Fig. 8. Cell-cell contact loss from the effects of OXO on peripheral actin filaments is correlated with wound healing abilities that are dependent on integrin 5, PKC, SFK and Akt/PKB activities. (A) Confluent RIE1 cells seeded onto 60 mm culture dishes were wounded, washed twice with 10% FBS-DMEM-H, and treated with DMSO or 10 µM OXO, as described in Materials and Methods. Phase contrast images were taken 17 or 24 hours after wounding and treatment. (B) Confluent cells in 60 mm culture dishes were wounded, washed, and then pretreated with LY294002 (LY, 20 µM), PP2 (10 µM), PP3 (10 µM) or ML9 (20 µM) 10 minutes before 10 µM OXO treatment. Another set of cells was treated with DMSO alone in parallel. After incubation for 20 hours, phase contrast images were taken. Note that ML9 treatment caused a certain level of cytotoxicity. (C) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Ad-cont), K376A DN PKC (Ad-DN PKC), or WT PKC (Ad-WT PKC) for 8 hours. After the viruses had been washed out, the cells were treated with DMSO or 10 µM OXO for 16 hours, and then images were taken of the wound area. Dotted lines indicate the starting lines for wound healing. Data shown are representative of three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Working model of cell-cell contact loss and wound healing through integrin 5-PKC signal transduction to regulate cofilin dephosphorylation and peripheral actin reorganization. When actin filaments were affected presumably by extracellular cues or reagent-mediated morphological changes, integrin 5-mediated PKC Ser643 phosphorylation may cause the activation of focal adhesion molecules including FAK and SFK. The PI3K/Akt pathway may be linked to PKC through a transmodulatory loop (refer to text). Activation of focal adhesion molecules and PI3K/Akt correlates with the dephosphorylation of cofilin Ser3 upon OXO treatment, presumably via the activation of cofilin phosphatase, SSH1L (Nishita et al., 2004) and via SSH1L activation-mediated inhibition of LIMK1 activity (Soosairajah et al., 2005). In particular, OXO treatment can also cause localization of nonphosphorylated cofilin at cell peripheries. These can result in severing of peripheral actin filaments via the dephosphorylated and thus activated cofilin. This dynamic peripheral actin reorganization may lead to cell-cell contact loss, and thereby enhance wound healing. However, specific signaling linkage(s) to dynamically regulate PKC, FAK, SFK, and PI3K/Akt activity, and actin reorganization, is impaired in cells expressing integrin 5 lacking its whole cytoplasmic tail (right side) which appears to be crucial for association with and signaling to PKC, although signaling for Erk1/2-mediated cell proliferation, probably via intact transmembrane and extracellular domains is still possible. Therefore, integrin-PKC-mediated dynamic regulation of cofilin activity and peripheral actin reorganization is responsible for cell-cell contact loss in response to stimuli that cause morphological changes.

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