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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 results in downmodulation of c-Flip expression. Exogenous expression of c-Flip protects HUVECs from anoikis and prevents caspase-8 activation in detached cells. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for 8 h, and the lysates were subjected to immunoblotting analysis by antibodies against c-Flip (top), caspase-8 (5F7; middle), and tubulin (bottom). (B) c-Flip expression was determined in attached cells or in cells that had been kept in suspension for the indicated times by immunoblot analysis (top two panels) and by RT-PCR (bottom three panels). As a control, protein expression levels in the lysates were analyzed by antitubulin immunoblotting (top), and mRNA expression levels by RT-PCR analysis with primers specific for GAPDH (bottom). The relative expression levels of Flip proteins as quantitated with a densitometric analysis are indicated. (C) Exogenous expression of c-Flip protects HUVECs from anoikis. HUVECs were cotransfected with an empty vector (control) or with plasmid encoding c-FlipL, together with plasmid coding for GFP. 24 h after transfection, the cells were either kept adherent or in suspension for 12 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. (D) Exogenous expression of c-Flip blocks caspase-8 activation in detached HUVECs. HUVECs were transiently transfected with an empty vector or with plasmid encoding c-FlipL. 24 h after transfection, the cells were kept adherent or in suspension for 12 h. In some of the experiments, the cells were simultaneously treated with 20 μM of the caspase-8 inhibitor z-IETD-fmk, as indicated. The cell lysates were analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme. The membrane was stripped and reprobed with an anti–α-tubulin control antibody to confirm equal loading (bottom).
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Figure 5: Cell detachment results in downmodulation of c-Flip expression. Exogenous expression of c-Flip protects HUVECs from anoikis and prevents caspase-8 activation in detached cells. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for 8 h, and the lysates were subjected to immunoblotting analysis by antibodies against c-Flip (top), caspase-8 (5F7; middle), and tubulin (bottom). (B) c-Flip expression was determined in attached cells or in cells that had been kept in suspension for the indicated times by immunoblot analysis (top two panels) and by RT-PCR (bottom three panels). As a control, protein expression levels in the lysates were analyzed by antitubulin immunoblotting (top), and mRNA expression levels by RT-PCR analysis with primers specific for GAPDH (bottom). The relative expression levels of Flip proteins as quantitated with a densitometric analysis are indicated. (C) Exogenous expression of c-Flip protects HUVECs from anoikis. HUVECs were cotransfected with an empty vector (control) or with plasmid encoding c-FlipL, together with plasmid coding for GFP. 24 h after transfection, the cells were either kept adherent or in suspension for 12 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. (D) Exogenous expression of c-Flip blocks caspase-8 activation in detached HUVECs. HUVECs were transiently transfected with an empty vector or with plasmid encoding c-FlipL. 24 h after transfection, the cells were kept adherent or in suspension for 12 h. In some of the experiments, the cells were simultaneously treated with 20 μM of the caspase-8 inhibitor z-IETD-fmk, as indicated. The cell lysates were analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme. The membrane was stripped and reprobed with an anti–α-tubulin control antibody to confirm equal loading (bottom).

Mentions: Previous reports have demonstrated that Fas-mediated apoptosis can be modulated at the level of caspase-8 activation by a mechanism that involves c-Flip, an endogenous caspase-8 inhibitor (Irmler et al. 1997), and it has been suggested that the balance between the expression levels of c-Flip and caspase-8 may control the sensitivity of a given cell to Fas-mediated killing (Scaffidi et al. 1999). Two different isoforms of c-Flip, namely Flip long (FlipL) and Flip short (FlipS), have been detected in various cell types and both isoforms are known to interfere with caspase-8 activation (Irmler et al. 1997; Scaffidi et al. 1999). As shown in Fig. 5 A, both FlipL and FlipS are expressed at relatively high levels compared with caspase-8 in adherent HUVECs. However, in HUVECs that have been detached for 8 h, the c-Flip protein levels are significantly reduced compared with caspase-8 levels (the apparent disappearance of the proform of caspase-8 protein is due to the processing of the protein that takes place during caspase-8 activation). To study the levels of c-Flip in detached HUVECs in detail, a time course analysis of the protein (Fig. 5 B, top) and mRNA levels (as measured by semiquantitative RT-PCR; Fig. 5 B, bottom) of FlipL and FlipS were carried out. Our findings indicate that HUVEC detachment induces a rapid reduction of FlipL and, more notably, FlipS at both mRNA and protein levels. A clear decrease in Flip protein can be detected as early as 6 h after cell detachment, demonstrating that the reduction in the c-Flip protein levels precedes the bulk of caspase-8 activation and anoikis observed in detached HUVECs. Thus, matrix attachment and detachment may control Fas-mediated apoptosis and caspase-8 activation by regulating the levels of the caspase-8 antagonist, c-Flip.


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 results in downmodulation of c-Flip expression. Exogenous expression of c-Flip protects HUVECs from anoikis and prevents caspase-8 activation in detached cells. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for 8 h, and the lysates were subjected to immunoblotting analysis by antibodies against c-Flip (top), caspase-8 (5F7; middle), and tubulin (bottom). (B) c-Flip expression was determined in attached cells or in cells that had been kept in suspension for the indicated times by immunoblot analysis (top two panels) and by RT-PCR (bottom three panels). As a control, protein expression levels in the lysates were analyzed by antitubulin immunoblotting (top), and mRNA expression levels by RT-PCR analysis with primers specific for GAPDH (bottom). The relative expression levels of Flip proteins as quantitated with a densitometric analysis are indicated. (C) Exogenous expression of c-Flip protects HUVECs from anoikis. HUVECs were cotransfected with an empty vector (control) or with plasmid encoding c-FlipL, together with plasmid coding for GFP. 24 h after transfection, the cells were either kept adherent or in suspension for 12 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. (D) Exogenous expression of c-Flip blocks caspase-8 activation in detached HUVECs. HUVECs were transiently transfected with an empty vector or with plasmid encoding c-FlipL. 24 h after transfection, the cells were kept adherent or in suspension for 12 h. In some of the experiments, the cells were simultaneously treated with 20 μM of the caspase-8 inhibitor z-IETD-fmk, as indicated. The cell lysates were analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme. The membrane was stripped and reprobed with an anti–α-tubulin control antibody to confirm equal loading (bottom).
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Figure 5: Cell detachment results in downmodulation of c-Flip expression. Exogenous expression of c-Flip protects HUVECs from anoikis and prevents caspase-8 activation in detached cells. (A) Cell lysates were prepared from adherent HUVECs and from HUVECs that had been kept in suspension for 8 h, and the lysates were subjected to immunoblotting analysis by antibodies against c-Flip (top), caspase-8 (5F7; middle), and tubulin (bottom). (B) c-Flip expression was determined in attached cells or in cells that had been kept in suspension for the indicated times by immunoblot analysis (top two panels) and by RT-PCR (bottom three panels). As a control, protein expression levels in the lysates were analyzed by antitubulin immunoblotting (top), and mRNA expression levels by RT-PCR analysis with primers specific for GAPDH (bottom). The relative expression levels of Flip proteins as quantitated with a densitometric analysis are indicated. (C) Exogenous expression of c-Flip protects HUVECs from anoikis. HUVECs were cotransfected with an empty vector (control) or with plasmid encoding c-FlipL, together with plasmid coding for GFP. 24 h after transfection, the cells were either kept adherent or in suspension for 12 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. (D) Exogenous expression of c-Flip blocks caspase-8 activation in detached HUVECs. HUVECs were transiently transfected with an empty vector or with plasmid encoding c-FlipL. 24 h after transfection, the cells were kept adherent or in suspension for 12 h. In some of the experiments, the cells were simultaneously treated with 20 μM of the caspase-8 inhibitor z-IETD-fmk, as indicated. The cell lysates were analyzed by immunoblotting with an anti–caspase-8 antibody (Ab-1) that detects the p18 active form of the enzyme. The membrane was stripped and reprobed with an anti–α-tubulin control antibody to confirm equal loading (bottom).
Mentions: Previous reports have demonstrated that Fas-mediated apoptosis can be modulated at the level of caspase-8 activation by a mechanism that involves c-Flip, an endogenous caspase-8 inhibitor (Irmler et al. 1997), and it has been suggested that the balance between the expression levels of c-Flip and caspase-8 may control the sensitivity of a given cell to Fas-mediated killing (Scaffidi et al. 1999). Two different isoforms of c-Flip, namely Flip long (FlipL) and Flip short (FlipS), have been detected in various cell types and both isoforms are known to interfere with caspase-8 activation (Irmler et al. 1997; Scaffidi et al. 1999). As shown in Fig. 5 A, both FlipL and FlipS are expressed at relatively high levels compared with caspase-8 in adherent HUVECs. However, in HUVECs that have been detached for 8 h, the c-Flip protein levels are significantly reduced compared with caspase-8 levels (the apparent disappearance of the proform of caspase-8 protein is due to the processing of the protein that takes place during caspase-8 activation). To study the levels of c-Flip in detached HUVECs in detail, a time course analysis of the protein (Fig. 5 B, top) and mRNA levels (as measured by semiquantitative RT-PCR; Fig. 5 B, bottom) of FlipL and FlipS were carried out. Our findings indicate that HUVEC detachment induces a rapid reduction of FlipL and, more notably, FlipS at both mRNA and protein levels. A clear decrease in Flip protein can be detected as early as 6 h after cell detachment, demonstrating that the reduction in the c-Flip protein levels precedes the bulk of caspase-8 activation and anoikis observed in detached HUVECs. Thus, matrix attachment and detachment may control Fas-mediated apoptosis and caspase-8 activation by regulating the levels of the caspase-8 antagonist, c-Flip.

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