QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 March 2008
doi: 10.1242/jcs.017194

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rygiel, T. P. Articles by Collard, J. G. Search for Related Content PubMed PubMed Citation Articles by Rygiel, T. P. Articles by Collard, J. G. Social Bookmarking                         What's this?

The Rac activator Tiam1 prevents keratinocyte apoptosis by controlling ROS-mediated ERK phosphorylation

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (59K): [in this window] [in a new window]   Fig. 1. Tiam1-KO keratinocytes are more susceptible to apoptosis than are wild-type (WT) keratinocytes. (A) Phase-contrast photographs of WT and Tiam1-deficient (KO) keratinocytes cultured on collagen IV-coated tissue culture plates. Cells were grown in normal culture conditions or heat shocked for 90 minutes at 43°C, followed by 6 hours recovery at 37°C. Bar, 50 µm. (B) Quantification of apoptosis induced by GF deprivation (24 hours) or heat-shock treatment in WT and Tiam1-KO keratinocytes. Both adherent and non-adherent cells were collected and subjected to DNA profile analysis. Cells with sub-G1 DNA content were considered to be apoptotic. Error bars represent standard deviation from triplicate samples. (C) Quantification of apoptosis induced by GF deprivation (24 hours) in WT and Tiam1-KO keratinocytes. Both adherent and non-adherent cells were collected, stained with annexin-V and the DNA stain propidium iodide (PI), and analyzed by FACS. Annexin-V-positive cells were considered to be apoptotic. Error bars represent standard deviation from three independent experiments. (D) Rac activity assay performed in lysates from WT and Tiam1-KO keratinocytes. Cells were GF starved for 30 minutes (GF–). Tiam1 and Rac were used as expression and loading controls, respectively. The data presented is a representative example of four independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Expression of Tiam1 rescues the apoptotic sensitivity of Tiam1-KO keratinocytes. (A) Immunoblot showing the expression of exogenous full-length Tiam1 in Tiam1-KO cells. E-cadherin was used as loading control. (B) Quantification of apoptosis upon GF deprivation for 24 hours by annexin-V staining and FACS analysis of Tiam1-KO keratinocytes expressing empty vector or exogenous Tiam1. Error bars represent standard deviation from three independent experiments. (C) Phase-contrast photographs of Tiam1-KO keratinocytes expressing exogenous Tiam1 in GF-containing medium and following GF starvation for 24 hours. Bar, 50 µm. (D) GF starvation (24 hours) induced apoptosis in WT keratinocytes transfected with shRNA constructs and in Tiam1-KO cells. The luciferase targeting sequence was used as a control shRNA (sh-Luc). Tiam1 downregulation was approximately 50%. Apoptosis was monitored by PARP cleavage and was seen only in GF-starved cells expressing Tiam1-specific siRNA or Tiam1-KO cells. (E) Western blot showing keratinocyte apoptosis (PARP cleavage) induced by GF starvation in WT, Tiam1-KO, and Tiam1-KO with exogenous expression of either full-length Tiam1 or an inactive mutant of Tiam1 (Tiam1-DH). Rac expression was used as loading control. (F) Estimation of apoptosis by quantification of free nucleosomes bound to DNA in WT, Tiam1-KO, and Tiam1-KO keratinocytes with exogenous expression of full-length Tiam1 or Tiam1-DH. Only the expression of full-length Tiam1 rescued apoptosis induced in Tiam1-KO keratinocytes by GF starvation. The presented data is a representative example of three independently performed experiments.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Tiam1-KO keratinocytes have impaired ERK phosphorylation – a necessary survival signal. (A) Quantification of apoptotic cells by annexin-V staining and subsequent FACS analysis. WT and Tiam1-KO keratinocytes were deprived of complete GF supplement and treated with EGF (20 ng/ml) or insulin (10 µg/ml) as indicated. Replacement of complete GF supplement by EGF or insulin rescued apoptosis. (B) Time-dependent induction of apoptosis and ERK1/2 phosphorylation upon GF starvation. Immunoblot shows PARP cleavage and ERK1/2 phosphorylation; Rac and total ERK are shown as loading controls. (C) Quantification of the data presented in B, normalized to total ERK2. (D) ERK signaling is required for survival upon GF starvation; insulin provides ERK1/2-independent survival signaling. WT and Tiam1-KO keratinocytes were cultured in absence of GF for 24 hours with or without EGF (20 ng/ml), insulin (10 µg/ml) or the MEK inhibitor PD-98059 (10 µM). The immunoblots shown are representative examples of three independent experiments. (E) Quantification of ERK1/2 phosphorylation from three independent experiments with conditions as in D. Values were normalized for total ERK and readouts corresponding to GF-supplemented conditions were set to 1. Error bars represent standard deviation. (F) Quantification of PARP cleaved product from three independent experiments with conditions as in D, values were normalized for total ERK. Error bars represent standard deviation. (G) Quantification of apoptosis induced by GF starvation, by annexin-V staining and FACS analysis. As for the experiment shown in D, WT and Tiam1-KO keratinocytes were cultured in absence of GFs for 24 hours with or without EGF (20 ng/ml), insulin (10 µg/ml) or the MEK inhibitor PD-98059 (10 µM). Error bars represent standard deviation from three independent experiments. (H) Western blot depicting phosphorylation of Akt and IB in WT and Tiam1-KO keratinocytes in control and GF-deprived conditions (4 hours). Total Akt and Rac are presented as loading controls.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Tiam1-KO keratinocytes have impaired ROS production. (A) The level of intracellular ROS in WT and Tiam1-KO keratinocytes was visualized by DCF staining. The cells were starved of GFs for 4 hours. Images were taken using an epifluorescence microscope. Bar, 50 µm. (B) Quantification of intracellular ROS in WT and Tiam1-KO keratinocytes upon GF starvation for 30 minutes and 4 hours. Cells were loaded with DCF, lysed and DCF-fluorescence was measured in the lysates. Fluorescence values were normalized for the amount of total protein present in the lysates. The fluorescence value of WT cells was set to 1. Error bars represent standard deviation from triplicate measurements. The result shown is a representative example of four independent experiments. (C) Quantification of ROS levels at a single-cell level in WT and Tiam1-KO keratinocytes by DCF staining and FACS analysis. (D) Western blot showing the exogenous expression of full-length Tiam1 and the deletion mutant of Tiam1 (Tiam1-DH) in Tiam1-KO keratinocytes. Expression of Rac is shown as a loading control. (E) Exogenous expression of full-length Tiam1 but not Tiam1-DH restores ROS production in Tiam1-KO keratinocytes. Intracellular ROS was measured as described in B. (F) Partial downregulation of Tiam1 reduces ROS production in WT keratinocytes, both in the presence and absence of GFs. Intracellular ROS content was measured as described in B. The data is a representative example of three independent experiments. (G) Immunoblot showing Tiam1 expression in WT keratinocytes upon shRNA expression. Partial downregulation of Tiam1 (50%) inhibits ERK1/2 phosphorylation (in GF-starved conditions) when compared with control cells. Tiam1 and total ERK were used as expression and loading controls, respectively.

View larger version (77K): [in this window] [in a new window]   Fig. 5. ROS and cell-matrix adhesions stimulate ERK phosphorylation in the absence of GFs. (A) Western blot depicting that ERK phosphorylation in GF-starved WT keratinocytes is dependent on the presence of ROS and negatively correlates with increasing concentrations of antioxidant (NAC). The cells were starved for 3 hours in the presence of different concentrations of the ROS scavenger NAC. Total ERK expression was used as loading control. (B) Immunoblot showing PARP cleavage and ERK phosphorylation in WT keratinocytes. The cells were cultured in the presence or absence of GFs and NAC, as indicated. PARP cleavage correlates with prolonged (20 hours) GF-starvation combined with NAC treatment. Rac was used as a loading control. NAC was added at the beginning of the experiment. (C) Phase-contrast images of WT keratinocytes subjected to GF starvation and/or NAC treatment. The images correspond to the conditions in B. Bar, 50 µm. The data presented are representative examples of three independent experiments. (D) Western blot showing ERK phosphorylation in WT and Tiam1-KO cells in adhesive control, GF-starved (3 hours) and suspension conditions. Total ERK serves as a loading control. Results are a typical example of three independent experiments. (E) Quantification of ERK activity from D, normalized for total ERK expression; readouts corresponding to GF-supplemented conditions were set to 1.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Nox-dependent ROS production controls ERK phosphorylation and survival. (A) The levels of intracellular ROS were quantified by DFC fluorescence measurement in cell lysates of WT and Tiam1-KO keratinocytes. WT and Tiam1-KO keratinocytes were cultured in the presence or absence of GFs for 4 hours and treated with the Nox inhibitor DPI (15 mM) in the last 30 minutes. Measurements were conducted as described in Fig. 4B. (B) Quantification of intracellular ROS levels in WT and Tiam1-KO keratinocytes treated with or without the antioxidant NAC (10 mM). NAC treatment was carried out for 30 minutes prior to analysis. (C) Quantification of intracellular ROS levels in WT and Tiam1-KO keratinocytes treated with or without the Nox inhibitor DPI (15 mM) or Apocynin (APO, 10 mg/ml) in the last 30 minutes. (D) Western blot showing expression of various Nox components (Nox1-Mox1, p67-phox, p47-phox and p22-phox) in WT and Tiam1-KO cells, Rac expression serves as loading control. (E) Immunoblot showing ERK phosphorylation in WT keratinocytes in control or in GF-deprived conditions (4 hours), in the presence or absence of NAC (10 mM) or DPI (15 µM). Total ERK and Rac expression were used as loading controls. The data are a representative example of two independently performed experiments. (F) Western blot showing PARP cleavage in WT keratinocytes GF-starved for 24 hours or heat shocked (90 minutes at 43°C). Cells were treated with NAC or DPI at the start of the experiment. Rac expression serves as a loading control. The figure is a representative example of four independently performed experiments. (G) Model of the regulation of cellular fate by survival and pro-apoptotic signaling. An apoptotic stimulus (e.g. GF starvation) induces the activation of Bcl2 pro-apoptotic proteins. Tiam1 acts in the survival pathway by controlling Rac-dependent Nox-mediated ROS production and ERK activation. GFs may provide survival signals independent of Tiam1-Rac signaling, leading to ERK and/or Akt or NFB activation.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter