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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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Exogenous expression of both Fas and caspase-8 is required to render attached HUVECs sensitive to Fas-mediated apoptosis. HUVECs were transfected with an empty vector (control), or with plasmids encoding Fas and procaspase-8, together with plasmid coding for the GFP. 24 h after transfection, the cells were stimulated or not for an additional 24 h with 1 μg/ml of anti-Fas antibody CH11. 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.
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Figure 6: Exogenous expression of both Fas and caspase-8 is required to render attached HUVECs sensitive to Fas-mediated apoptosis. HUVECs were transfected with an empty vector (control), or with plasmids encoding Fas and procaspase-8, together with plasmid coding for the GFP. 24 h after transfection, the cells were stimulated or not for an additional 24 h with 1 μg/ml of anti-Fas antibody CH11. 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.

Mentions: We then examined the expression levels of Fas in attached and detached HUVECs. As reported previously, HUVECs cultured as a monolayer express detectable levels of Fas (Fig. 4 A). Detachment of HUVECs results in a threefold increase in the cell surface levels of Fas by 12 h after detachment (Fig. 4 A). The detachment-induced increase in Fas expression can be detected also at the mRNA level. A semiquantitative reverse transcriptase (RT)-PCR analysis of Fas mRNA expression indicates that cell detachment induces an increase in Fas mRNA levels as early as in 3 h, with a peak in the induction of Fas mRNA levels occurring 6 h after cell detachment (Fig. 4 B). To examine whether matrix attachment protects endothelial cells from Fas-mediated apoptosis by downregulating the cell surface levels of Fas, attached HUVECs were transiently transfected with a plasmid coding for Fas. In several independent experiments, transient transfection resulted, on average, in a two- to threefold increase in the cell surface levels of Fas (not shown). However, ectopic expression of Fas did not result in an induction of apoptosis in attached HUVECs, and neither did it sensitize the cells to CH11-mediated killing (see Fig. 6). Thus, although upregulation of Fas may be necessary for anoikis and Fas-mediated apoptosis to take place in detached cells, enhanced Fas expression alone is not sufficient to sensitize adherent HUVECs for Fas-mediated killing. Upon examination of DISC formation and caspase-8 activation in Fas-transfected, CH11-treated HUVECs, we observed Fas–FADD interaction but no caspase-8 activation (not shown). Taken together, these results suggest that matrix attachment regulates (at least) one additional step in Fas signaling, which takes place downstream of DISC formation but upstream or at the level of caspase-8 activation.


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)

Exogenous expression of both Fas and caspase-8 is required to render attached HUVECs sensitive to Fas-mediated apoptosis. HUVECs were transfected with an empty vector (control), or with plasmids encoding Fas and procaspase-8, together with plasmid coding for the GFP. 24 h after transfection, the cells were stimulated or not for an additional 24 h with 1 μg/ml of anti-Fas antibody CH11. 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196007&req=5

Figure 6: Exogenous expression of both Fas and caspase-8 is required to render attached HUVECs sensitive to Fas-mediated apoptosis. HUVECs were transfected with an empty vector (control), or with plasmids encoding Fas and procaspase-8, together with plasmid coding for the GFP. 24 h after transfection, the cells were stimulated or not for an additional 24 h with 1 μg/ml of anti-Fas antibody CH11. 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.
Mentions: We then examined the expression levels of Fas in attached and detached HUVECs. As reported previously, HUVECs cultured as a monolayer express detectable levels of Fas (Fig. 4 A). Detachment of HUVECs results in a threefold increase in the cell surface levels of Fas by 12 h after detachment (Fig. 4 A). The detachment-induced increase in Fas expression can be detected also at the mRNA level. A semiquantitative reverse transcriptase (RT)-PCR analysis of Fas mRNA expression indicates that cell detachment induces an increase in Fas mRNA levels as early as in 3 h, with a peak in the induction of Fas mRNA levels occurring 6 h after cell detachment (Fig. 4 B). To examine whether matrix attachment protects endothelial cells from Fas-mediated apoptosis by downregulating the cell surface levels of Fas, attached HUVECs were transiently transfected with a plasmid coding for Fas. In several independent experiments, transient transfection resulted, on average, in a two- to threefold increase in the cell surface levels of Fas (not shown). However, ectopic expression of Fas did not result in an induction of apoptosis in attached HUVECs, and neither did it sensitize the cells to CH11-mediated killing (see Fig. 6). Thus, although upregulation of Fas may be necessary for anoikis and Fas-mediated apoptosis to take place in detached cells, enhanced Fas expression alone is not sufficient to sensitize adherent HUVECs for Fas-mediated killing. Upon examination of DISC formation and caspase-8 activation in Fas-transfected, CH11-treated HUVECs, we observed Fas–FADD interaction but no caspase-8 activation (not shown). Taken together, these results suggest that matrix attachment regulates (at least) one additional step in Fas signaling, which takes place downstream of DISC formation but upstream or at the level of caspase-8 activation.

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