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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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Cell detachment induces Fas/Fas-L interaction, DISC formation, and caspase-8 activation in HUVECs. Inhibition of anoikis by dnFADD and caspase-8 inhibitors. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for the indicated times, and the lysates were subjected to immunoprecipitation analysis by anti–Fas-L antibodies (clone 33). Immunoprecipitates (IP) were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top). The membranes were stripped and reprobed with anti–Fas-L antibodies (clone 33) to confirm equal amounts of Fas-L in the precipitates (bottom). Similar results were obtained when anti–Fas-L antibody G247-4 was used (data not shown). (B) The cell lysates were subjected to immunoprecipitations by anti-FADD antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top), and the membranes were stripped and reprobed with anti-FADD antibodies to confirm equal amounts of FADD in the precipitates (bottom). C, Control cell lysate prepared from Jurkat T cells that had been activated by T cell receptor stimulation. (C) HUVECs were transiently transfected with an empty vector (control) or with an expression plasmid encoding dnFADD, together with plasmid coding for the green fluorescent protein (GFP). 24 h after transfection, the cells were either kept adherent or kept in suspension for the indicated time periods. The cells were then washed and propidium iodide was added for 20 min on ice. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP. In a separate experiment, we monitored apoptosis by staining the cells with Hoechst dye and found that results obtained with propidium iodide and Hoechst were indistinguishable from each other (not shown). Thus, the results obtained with the Hoechst dye confirm that cell death observed under our experimental conditions results from apoptosis, rather than necrosis. (D) HUVECs were left adherent or kept in suspension for 12 h in the presence or absence of the indicated concentrations of caspase-8 inhibitor z-IETD-fmk. In the left panel, apoptosis was determined by DNA fragmentation analysis; bars indicate SD in a representative experiment done in triplicate. In the right panel, cell lysates were prepared and analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme.
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Figure 3: Cell detachment induces Fas/Fas-L interaction, DISC formation, and caspase-8 activation in HUVECs. Inhibition of anoikis by dnFADD and caspase-8 inhibitors. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for the indicated times, and the lysates were subjected to immunoprecipitation analysis by anti–Fas-L antibodies (clone 33). Immunoprecipitates (IP) were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top). The membranes were stripped and reprobed with anti–Fas-L antibodies (clone 33) to confirm equal amounts of Fas-L in the precipitates (bottom). Similar results were obtained when anti–Fas-L antibody G247-4 was used (data not shown). (B) The cell lysates were subjected to immunoprecipitations by anti-FADD antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top), and the membranes were stripped and reprobed with anti-FADD antibodies to confirm equal amounts of FADD in the precipitates (bottom). C, Control cell lysate prepared from Jurkat T cells that had been activated by T cell receptor stimulation. (C) HUVECs were transiently transfected with an empty vector (control) or with an expression plasmid encoding dnFADD, together with plasmid coding for the green fluorescent protein (GFP). 24 h after transfection, the cells were either kept adherent or kept in suspension for the indicated time periods. The cells were then washed and propidium iodide was added for 20 min on ice. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP. In a separate experiment, we monitored apoptosis by staining the cells with Hoechst dye and found that results obtained with propidium iodide and Hoechst were indistinguishable from each other (not shown). Thus, the results obtained with the Hoechst dye confirm that cell death observed under our experimental conditions results from apoptosis, rather than necrosis. (D) HUVECs were left adherent or kept in suspension for 12 h in the presence or absence of the indicated concentrations of caspase-8 inhibitor z-IETD-fmk. In the left panel, apoptosis was determined by DNA fragmentation analysis; bars indicate SD in a representative experiment done in triplicate. In the right panel, cell lysates were prepared and analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme.

Mentions: To further examine the role of the Fas/Fas-L pathway in anoikis, we undertook several biochemical approaches to study Fas signaling in detached endothelial cells. First, we examined whether cell detachment induces Fas/Fas-L interaction in HUVECs. As shown in Fig. 3 A, Fas becomes associated with Fas-L after detachment of HUVECs, and this association precedes induction of the bulk of anoikis in these cells (see Fig. 1 A). Thus, Fas/Fas-L association can be clearly detected 6 h after cell detachment, the interaction peaking 12 h after detachment of the cells. Importantly, the molecular mass of Fas detected in the Fas-L immunoprecipitates is 110 kD, which corresponds to the oligomerized form of Fas that is induced after binding of Fas-L to Fas (Kamitani et al. 1997).


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)

Cell detachment induces Fas/Fas-L interaction, DISC formation, and caspase-8 activation in HUVECs. Inhibition of anoikis by dnFADD and caspase-8 inhibitors. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for the indicated times, and the lysates were subjected to immunoprecipitation analysis by anti–Fas-L antibodies (clone 33). Immunoprecipitates (IP) were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top). The membranes were stripped and reprobed with anti–Fas-L antibodies (clone 33) to confirm equal amounts of Fas-L in the precipitates (bottom). Similar results were obtained when anti–Fas-L antibody G247-4 was used (data not shown). (B) The cell lysates were subjected to immunoprecipitations by anti-FADD antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top), and the membranes were stripped and reprobed with anti-FADD antibodies to confirm equal amounts of FADD in the precipitates (bottom). C, Control cell lysate prepared from Jurkat T cells that had been activated by T cell receptor stimulation. (C) HUVECs were transiently transfected with an empty vector (control) or with an expression plasmid encoding dnFADD, together with plasmid coding for the green fluorescent protein (GFP). 24 h after transfection, the cells were either kept adherent or kept in suspension for the indicated time periods. The cells were then washed and propidium iodide was added for 20 min on ice. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP. In a separate experiment, we monitored apoptosis by staining the cells with Hoechst dye and found that results obtained with propidium iodide and Hoechst were indistinguishable from each other (not shown). Thus, the results obtained with the Hoechst dye confirm that cell death observed under our experimental conditions results from apoptosis, rather than necrosis. (D) HUVECs were left adherent or kept in suspension for 12 h in the presence or absence of the indicated concentrations of caspase-8 inhibitor z-IETD-fmk. In the left panel, apoptosis was determined by DNA fragmentation analysis; bars indicate SD in a representative experiment done in triplicate. In the right panel, cell lysates were prepared and analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme.
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Figure 3: Cell detachment induces Fas/Fas-L interaction, DISC formation, and caspase-8 activation in HUVECs. Inhibition of anoikis by dnFADD and caspase-8 inhibitors. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for the indicated times, and the lysates were subjected to immunoprecipitation analysis by anti–Fas-L antibodies (clone 33). Immunoprecipitates (IP) were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top). The membranes were stripped and reprobed with anti–Fas-L antibodies (clone 33) to confirm equal amounts of Fas-L in the precipitates (bottom). Similar results were obtained when anti–Fas-L antibody G247-4 was used (data not shown). (B) The cell lysates were subjected to immunoprecipitations by anti-FADD antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-Fas antibodies (clone 13; top), and the membranes were stripped and reprobed with anti-FADD antibodies to confirm equal amounts of FADD in the precipitates (bottom). C, Control cell lysate prepared from Jurkat T cells that had been activated by T cell receptor stimulation. (C) HUVECs were transiently transfected with an empty vector (control) or with an expression plasmid encoding dnFADD, together with plasmid coding for the green fluorescent protein (GFP). 24 h after transfection, the cells were either kept adherent or kept in suspension for the indicated time periods. The cells were then washed and propidium iodide was added for 20 min on ice. Apoptosis analysis by FACS® was carried out in the double positive cell population for propidium iodide and fluorescent GFP. In a separate experiment, we monitored apoptosis by staining the cells with Hoechst dye and found that results obtained with propidium iodide and Hoechst were indistinguishable from each other (not shown). Thus, the results obtained with the Hoechst dye confirm that cell death observed under our experimental conditions results from apoptosis, rather than necrosis. (D) HUVECs were left adherent or kept in suspension for 12 h in the presence or absence of the indicated concentrations of caspase-8 inhibitor z-IETD-fmk. In the left panel, apoptosis was determined by DNA fragmentation analysis; bars indicate SD in a representative experiment done in triplicate. In the right panel, cell lysates were prepared and analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme.
Mentions: To further examine the role of the Fas/Fas-L pathway in anoikis, we undertook several biochemical approaches to study Fas signaling in detached endothelial cells. First, we examined whether cell detachment induces Fas/Fas-L interaction in HUVECs. As shown in Fig. 3 A, Fas becomes associated with Fas-L after detachment of HUVECs, and this association precedes induction of the bulk of anoikis in these cells (see Fig. 1 A). Thus, Fas/Fas-L association can be clearly detected 6 h after cell detachment, the interaction peaking 12 h after detachment of the cells. Importantly, the molecular mass of Fas detected in the Fas-L immunoprecipitates is 110 kD, which corresponds to the oligomerized form of Fas that is induced after binding of Fas-L to Fas (Kamitani et al. 1997).

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