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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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Detachment-induced apoptosis in HUVECs is Fas/Fas-L dependent. HUVECs were detached and either kept in suspension or on polyhema-coated dishes in complete growth medium (EGM medium containing 2% FCS, 10 ng/ml hEGF, and 1 μg/ml hydrocortisone, supplemented with 12 μg/ml of bovine brain extract, 2 mM l-glutamine, 50 μg/ml streptomycin, and 50 U/ml penicillin) for the indicated time periods, and cell death was determined by (A) trypan blue exclusion or (B) DNA fragmentation analysis. Identical results were obtained under the two experimental conditions; results shown are for cells kept in suspension, and these experimental conditions were used in all subsequent experiments involving detachment-induced cell death. (C) HUVECs were kept adherent or in suspension for 12 h in the presence or absence of 5 μg/ml of inhibitory anti–Fas-L antibodies (NOK-2), inhibitory anti-Fas antibodies (ZB4), or with control antibodies (IgG). Apoptosis was determined by DNA fragmentation analysis. In A and B, results are shown for representative experiments independently carried out three times. In C, bars indicate SD in a representative experiment done in triplicate.
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Figure 1: Detachment-induced apoptosis in HUVECs is Fas/Fas-L dependent. HUVECs were detached and either kept in suspension or on polyhema-coated dishes in complete growth medium (EGM medium containing 2% FCS, 10 ng/ml hEGF, and 1 μg/ml hydrocortisone, supplemented with 12 μg/ml of bovine brain extract, 2 mM l-glutamine, 50 μg/ml streptomycin, and 50 U/ml penicillin) for the indicated time periods, and cell death was determined by (A) trypan blue exclusion or (B) DNA fragmentation analysis. Identical results were obtained under the two experimental conditions; results shown are for cells kept in suspension, and these experimental conditions were used in all subsequent experiments involving detachment-induced cell death. (C) HUVECs were kept adherent or in suspension for 12 h in the presence or absence of 5 μg/ml of inhibitory anti–Fas-L antibodies (NOK-2), inhibitory anti-Fas antibodies (ZB4), or with control antibodies (IgG). Apoptosis was determined by DNA fragmentation analysis. In A and B, results are shown for representative experiments independently carried out three times. In C, bars indicate SD in a representative experiment done in triplicate.

Mentions: Previous studies have indicated that HUVECs undergo anoikis when denied matrix attachment (Meredith et al. 1993; Re et al. 1994), and we found the same here. As shown in Fig. 1 A, endothelial cell viability decreases by 75% over a 48-h time period after cell detachment in the presence of serum, and a clear induction (25%) in cell death is observed 12 h after detachment of the cells. The loss of cell viability is a result of apoptosis as attested by the significant induction in DNA fragmentation, a hallmark of apoptotic death, after cell detachment (Fig. 1 B). To examine whether anoikis in HUVECs involves activation of the Fas/Fas-L pathway, we used inhibitory antibodies against Fas and Fas-L that have been shown previously to suppress the Fas/Fas-L pathway and inhibit Fas-mediated apoptosis (Kayagaki et al. 1995; Srivastava et al. 1999). As shown in Fig. 1 C, treatment of detached HUVECs with anti–Fas-L antibodies inhibits anoikis in these cells by ∼60%. Inhibitory anti-Fas antibodies, alone or in combination with the anti–Fas-L antibodies, also significantly block anoikis in HUVECs (Fig. 1 C). Several irrelevant isotype–matched control antibodies failed to have any effect on cell survival or anoikis in HUVECs (Fig. 1 C). AICD in T lymphocytes is known to take place via the Fas/Fas-L pathway, and as a control, we found that AICD in Jurkat cells can similarly be blocked by ∼70% with anti–Fas-L or anti-Fas antibodies (not shown; Aoudjit and Vuori 2000). These antibodies did not have any effect on serum withdrawal–induced apoptosis in endothelial cells, which occurs in a Fas-independent manner (not shown). Thus, these results suggest that activation of Fas by its ligand, Fas-L, has a significant functional role in the induction of anoikis in HUVECs.


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)

Detachment-induced apoptosis in HUVECs is Fas/Fas-L dependent. HUVECs were detached and either kept in suspension or on polyhema-coated dishes in complete growth medium (EGM medium containing 2% FCS, 10 ng/ml hEGF, and 1 μg/ml hydrocortisone, supplemented with 12 μg/ml of bovine brain extract, 2 mM l-glutamine, 50 μg/ml streptomycin, and 50 U/ml penicillin) for the indicated time periods, and cell death was determined by (A) trypan blue exclusion or (B) DNA fragmentation analysis. Identical results were obtained under the two experimental conditions; results shown are for cells kept in suspension, and these experimental conditions were used in all subsequent experiments involving detachment-induced cell death. (C) HUVECs were kept adherent or in suspension for 12 h in the presence or absence of 5 μg/ml of inhibitory anti–Fas-L antibodies (NOK-2), inhibitory anti-Fas antibodies (ZB4), or with control antibodies (IgG). Apoptosis was determined by DNA fragmentation analysis. In A and B, results are shown for representative experiments independently carried out three times. In C, 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 1: Detachment-induced apoptosis in HUVECs is Fas/Fas-L dependent. HUVECs were detached and either kept in suspension or on polyhema-coated dishes in complete growth medium (EGM medium containing 2% FCS, 10 ng/ml hEGF, and 1 μg/ml hydrocortisone, supplemented with 12 μg/ml of bovine brain extract, 2 mM l-glutamine, 50 μg/ml streptomycin, and 50 U/ml penicillin) for the indicated time periods, and cell death was determined by (A) trypan blue exclusion or (B) DNA fragmentation analysis. Identical results were obtained under the two experimental conditions; results shown are for cells kept in suspension, and these experimental conditions were used in all subsequent experiments involving detachment-induced cell death. (C) HUVECs were kept adherent or in suspension for 12 h in the presence or absence of 5 μg/ml of inhibitory anti–Fas-L antibodies (NOK-2), inhibitory anti-Fas antibodies (ZB4), or with control antibodies (IgG). Apoptosis was determined by DNA fragmentation analysis. In A and B, results are shown for representative experiments independently carried out three times. In C, bars indicate SD in a representative experiment done in triplicate.
Mentions: Previous studies have indicated that HUVECs undergo anoikis when denied matrix attachment (Meredith et al. 1993; Re et al. 1994), and we found the same here. As shown in Fig. 1 A, endothelial cell viability decreases by 75% over a 48-h time period after cell detachment in the presence of serum, and a clear induction (25%) in cell death is observed 12 h after detachment of the cells. The loss of cell viability is a result of apoptosis as attested by the significant induction in DNA fragmentation, a hallmark of apoptotic death, after cell detachment (Fig. 1 B). To examine whether anoikis in HUVECs involves activation of the Fas/Fas-L pathway, we used inhibitory antibodies against Fas and Fas-L that have been shown previously to suppress the Fas/Fas-L pathway and inhibit Fas-mediated apoptosis (Kayagaki et al. 1995; Srivastava et al. 1999). As shown in Fig. 1 C, treatment of detached HUVECs with anti–Fas-L antibodies inhibits anoikis in these cells by ∼60%. Inhibitory anti-Fas antibodies, alone or in combination with the anti–Fas-L antibodies, also significantly block anoikis in HUVECs (Fig. 1 C). Several irrelevant isotype–matched control antibodies failed to have any effect on cell survival or anoikis in HUVECs (Fig. 1 C). AICD in T lymphocytes is known to take place via the Fas/Fas-L pathway, and as a control, we found that AICD in Jurkat cells can similarly be blocked by ∼70% with anti–Fas-L or anti-Fas antibodies (not shown; Aoudjit and Vuori 2000). These antibodies did not have any effect on serum withdrawal–induced apoptosis in endothelial cells, which occurs in a Fas-independent manner (not shown). Thus, these results suggest that activation of Fas by its ligand, Fas-L, has a significant functional role in the induction of anoikis in HUVECs.

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