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Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis.

Aoudjit F, Vuori K - J. Cell Biol. (2001)

Bottom Line: The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression.We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L.Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.

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The Erk pathway functions as a survival pathway in attached HUVECs and modulates c-Flip expression. (A) Exogenous expression of activated Raf-1 enhances c-Flip expression in detached HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 24 h. c-Flip expression was determined by immunoblot analysis (top) and by RT-PCR (bottom). (B) Cell detachment results in the inactivation of the MAPK/Erk pathway in a time-dependent manner. HUVECs were kept adherent or in suspension for the indicated times. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom). As indicated, in one of the experiments, the effectiveness of the MEK inhibitor PD98059 was analyzed by treating adherent HUVECs for 16 h before cell lysis. (C) Inhibition of the Erk pathway in adherent HUVECs downregulates c-Flip expression. HUVECs were treated or not with the MEK inhibitor PD98059 for 16 h and c-Flip expression was determined by immunoblotting (top) and by RT-PCR (bottom). (D) Inhibition of the Erk pathway in adherent HUVECs sensitizes the cells to Fas-mediated apoptosis. HUVECs were transiently transfected with an empty control vector or with a plasmid encoding Fas, together with plasmid coding for GFP. After transfection, the cells were treated or not with 25 μM of the MEK inhibitor PD98059 in the presence or absence of 1 μg/ml of the anti-Fas antibody CH11 for 24 h. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP as described above. Bars indicate SD in a representative experiment done in triplicate. (E) Exogenous expression of activated Raf-1 enhances Erk phosphorylation in HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 12 h. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom).
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Figure 8: The Erk pathway functions as a survival pathway in attached HUVECs and modulates c-Flip expression. (A) Exogenous expression of activated Raf-1 enhances c-Flip expression in detached HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 24 h. c-Flip expression was determined by immunoblot analysis (top) and by RT-PCR (bottom). (B) Cell detachment results in the inactivation of the MAPK/Erk pathway in a time-dependent manner. HUVECs were kept adherent or in suspension for the indicated times. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom). As indicated, in one of the experiments, the effectiveness of the MEK inhibitor PD98059 was analyzed by treating adherent HUVECs for 16 h before cell lysis. (C) Inhibition of the Erk pathway in adherent HUVECs downregulates c-Flip expression. HUVECs were treated or not with the MEK inhibitor PD98059 for 16 h and c-Flip expression was determined by immunoblotting (top) and by RT-PCR (bottom). (D) Inhibition of the Erk pathway in adherent HUVECs sensitizes the cells to Fas-mediated apoptosis. HUVECs were transiently transfected with an empty control vector or with a plasmid encoding Fas, together with plasmid coding for GFP. After transfection, the cells were treated or not with 25 μM of the MEK inhibitor PD98059 in the presence or absence of 1 μg/ml of the anti-Fas antibody CH11 for 24 h. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP as described above. Bars indicate SD in a representative experiment done in triplicate. (E) Exogenous expression of activated Raf-1 enhances Erk phosphorylation in HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 12 h. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom).

Mentions: In many cell types, matrix attachment has been shown to result in activation of the PI 3′-kinase/Akt and MAPK/Erk pathways, whereas cell detachment is known to downregulate these activities (for review see Giancotti and Ruoslahti 1999; Schlaepfer et al. 1999). Furthermore, activation of the PI 3′-kinase/Akt and the Erk pathways has been shown to protect MDCK and CCL39 cells, respectively, against detachment-induced cell death (Khwaja et al. 1997; Le Gall et al. 2000). We found that matrix attachment activates and matrix detachment inactivates the two pathways in HUVECs (results for the Erk pathway are shown in Fig. 8 B), and we therefore decided to examine the role of these two survival pathways in adhesion-mediated protection against Fas-induced apoptosis and anoikis in endothelial cells.


Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis.

Aoudjit F, Vuori K - J. Cell Biol. (2001)

The Erk pathway functions as a survival pathway in attached HUVECs and modulates c-Flip expression. (A) Exogenous expression of activated Raf-1 enhances c-Flip expression in detached HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 24 h. c-Flip expression was determined by immunoblot analysis (top) and by RT-PCR (bottom). (B) Cell detachment results in the inactivation of the MAPK/Erk pathway in a time-dependent manner. HUVECs were kept adherent or in suspension for the indicated times. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom). As indicated, in one of the experiments, the effectiveness of the MEK inhibitor PD98059 was analyzed by treating adherent HUVECs for 16 h before cell lysis. (C) Inhibition of the Erk pathway in adherent HUVECs downregulates c-Flip expression. HUVECs were treated or not with the MEK inhibitor PD98059 for 16 h and c-Flip expression was determined by immunoblotting (top) and by RT-PCR (bottom). (D) Inhibition of the Erk pathway in adherent HUVECs sensitizes the cells to Fas-mediated apoptosis. HUVECs were transiently transfected with an empty control vector or with a plasmid encoding Fas, together with plasmid coding for GFP. After transfection, the cells were treated or not with 25 μM of the MEK inhibitor PD98059 in the presence or absence of 1 μg/ml of the anti-Fas antibody CH11 for 24 h. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP as described above. Bars indicate SD in a representative experiment done in triplicate. (E) Exogenous expression of activated Raf-1 enhances Erk phosphorylation in HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 12 h. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom).
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Figure 8: The Erk pathway functions as a survival pathway in attached HUVECs and modulates c-Flip expression. (A) Exogenous expression of activated Raf-1 enhances c-Flip expression in detached HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 24 h. c-Flip expression was determined by immunoblot analysis (top) and by RT-PCR (bottom). (B) Cell detachment results in the inactivation of the MAPK/Erk pathway in a time-dependent manner. HUVECs were kept adherent or in suspension for the indicated times. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom). As indicated, in one of the experiments, the effectiveness of the MEK inhibitor PD98059 was analyzed by treating adherent HUVECs for 16 h before cell lysis. (C) Inhibition of the Erk pathway in adherent HUVECs downregulates c-Flip expression. HUVECs were treated or not with the MEK inhibitor PD98059 for 16 h and c-Flip expression was determined by immunoblotting (top) and by RT-PCR (bottom). (D) Inhibition of the Erk pathway in adherent HUVECs sensitizes the cells to Fas-mediated apoptosis. HUVECs were transiently transfected with an empty control vector or with a plasmid encoding Fas, together with plasmid coding for GFP. After transfection, the cells were treated or not with 25 μM of the MEK inhibitor PD98059 in the presence or absence of 1 μg/ml of the anti-Fas antibody CH11 for 24 h. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP as described above. Bars indicate SD in a representative experiment done in triplicate. (E) Exogenous expression of activated Raf-1 enhances Erk phosphorylation in HUVECs. HUVECs were transfected with an empty vector (Control) or with a plasmid encoding activated form of c-Raf-1 (Raf-CAAX). After transfection, the cells were kept adherent or in suspension for 12 h. Cell lysates were prepared and Erk activation was determined by an immunoblot analysis with an anti–phospho-Erk1/2 antibody (top). The membrane was stripped and reprobed with an anti-Erk2 antibody to confirm equal loading (bottom).
Mentions: In many cell types, matrix attachment has been shown to result in activation of the PI 3′-kinase/Akt and MAPK/Erk pathways, whereas cell detachment is known to downregulate these activities (for review see Giancotti and Ruoslahti 1999; Schlaepfer et al. 1999). Furthermore, activation of the PI 3′-kinase/Akt and the Erk pathways has been shown to protect MDCK and CCL39 cells, respectively, against detachment-induced cell death (Khwaja et al. 1997; Le Gall et al. 2000). We found that matrix attachment activates and matrix detachment inactivates the two pathways in HUVECs (results for the Erk pathway are shown in Fig. 8 B), and we therefore decided to examine the role of these two survival pathways in adhesion-mediated protection against Fas-induced apoptosis and anoikis in endothelial cells.

Bottom Line: The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression.We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L.Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.

Show MeSH
Related in: MedlinePlus