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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 sensitizes HUVECs to Fas-mediated apoptosis. The ability of agonistic anti-Fas antibody (CH11) to induce apoptosis was analyzed in attached and detached HUVECs. The cells were incubated with or without 1 μg/ml of CH11 for 12 h in the presence or absence of blocking anti–Fas-L antibody (NOK-2), and apoptosis was determined by DNA fragmentation analysis. Bars indicate SD in a representative experiment done in triplicate.
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Figure 2: Cell detachment sensitizes HUVECs to Fas-mediated apoptosis. The ability of agonistic anti-Fas antibody (CH11) to induce apoptosis was analyzed in attached and detached HUVECs. The cells were incubated with or without 1 μg/ml of CH11 for 12 h in the presence or absence of blocking anti–Fas-L antibody (NOK-2), and apoptosis was determined by DNA fragmentation analysis. Bars indicate SD in a representative experiment done in triplicate.

Mentions: To examine whether matrix attachment in turn might protect HUVECs against Fas-mediated apoptosis, adherent HUVECs were treated with an agonistic Fas-antibody, CH11, to induce activation of Fas signaling. We found that adherent HUVECs are highly resistant to Fas-mediated killing (Fig. 2). Upon cell detachment, HUVECs undergo programmed cell death, as expected, as a result of anoikis. However, addition of CH11 to detached cells further enhances cell death, suggesting that cell detachment sensitizes HUVECs to Fas-mediated apoptosis (Fig. 2). We used the inhibitory anti–Fas-L antibody to block Fas-L–dependent induction of Fas signaling in detached cells, and studied the effect of CH11 under these conditions. As shown in Fig. 2, a significant amount of apoptosis takes place in detached, CH11-treated HUVECs, in which Fas-L–induced apoptosis has been eliminated by the inhibitory anti–Fas-L antibody. Thus, cell detachment not only induces activation of the Fas pathway by Fas-L, but also sensitizes endothelial cells to activation of Fas-mediated apoptosis by exogenously added Fas agonists.


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 sensitizes HUVECs to Fas-mediated apoptosis. The ability of agonistic anti-Fas antibody (CH11) to induce apoptosis was analyzed in attached and detached HUVECs. The cells were incubated with or without 1 μg/ml of CH11 for 12 h in the presence or absence of blocking anti–Fas-L antibody (NOK-2), and apoptosis was determined by DNA fragmentation analysis. Bars indicate SD in a representative experiment done in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196007&req=5

Figure 2: Cell detachment sensitizes HUVECs to Fas-mediated apoptosis. The ability of agonistic anti-Fas antibody (CH11) to induce apoptosis was analyzed in attached and detached HUVECs. The cells were incubated with or without 1 μg/ml of CH11 for 12 h in the presence or absence of blocking anti–Fas-L antibody (NOK-2), and apoptosis was determined by DNA fragmentation analysis. Bars indicate SD in a representative experiment done in triplicate.
Mentions: To examine whether matrix attachment in turn might protect HUVECs against Fas-mediated apoptosis, adherent HUVECs were treated with an agonistic Fas-antibody, CH11, to induce activation of Fas signaling. We found that adherent HUVECs are highly resistant to Fas-mediated killing (Fig. 2). Upon cell detachment, HUVECs undergo programmed cell death, as expected, as a result of anoikis. However, addition of CH11 to detached cells further enhances cell death, suggesting that cell detachment sensitizes HUVECs to Fas-mediated apoptosis (Fig. 2). We used the inhibitory anti–Fas-L antibody to block Fas-L–dependent induction of Fas signaling in detached cells, and studied the effect of CH11 under these conditions. As shown in Fig. 2, a significant amount of apoptosis takes place in detached, CH11-treated HUVECs, in which Fas-L–induced apoptosis has been eliminated by the inhibitory anti–Fas-L antibody. Thus, cell detachment not only induces activation of the Fas pathway by Fas-L, but also sensitizes endothelial cells to activation of Fas-mediated apoptosis by exogenously added Fas agonists.

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