Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis - PubMed (original) (raw)

Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis

D Ilić et al. J Cell Biol. 1998.

Abstract

In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C lambda/iota and cytosolic phospholipase A2. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.

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Figures

Figure 1

FAK suppresses a p53-regulated apoptosis pathway in embryoid bodies and endothelial cells. (A) Withdrawal of serum induces apoptosis in FAK− EB generated from wild-type and FAK− TT2 ES cells (Ilić et al., 1995_a_). Cells were cultured for 11 d without leukemia inhibitory factor and in the presence of serum to initiate differentiation. They were then cultured for an additional 24 h either with (+), or without (−) serum. The presence of apoptotic cells was assessed by TUNEL staining, and nuclei were visualized with Hoechst as described in the Materials and Methods. (B) p53 deficiency permits survival, despite the absence of FAK, in anchorage-dependent, serum-deprived endothelial cells. Endothelial cells were isolated from FAK+ or FAK− EB or embryos and immortalized by pmT or incorporation of mutated p53, as described in the Materials and Methods. Apoptosis was assessed by a flow cytometric assay: PI, propidium iodide; Hoechst, Hoechst 33342. In each plot, the events represented in red (upper gated area) correspond to the PI/Hoechst ratios characteristic of an apoptotic population, whereas the events represented in green (lower gated area) correspond to nonapoptotic cells (Hamel et al., 1996). Endothelial cell lines were cultured for 48 h in the presence of serum and for an additional 24 h in the presence (+) or absence (−) of serum. All endothelial lines cultured in the presence of serum, as well as both p53-deficient cell lines cultured without serum, displayed low levels of apoptosis. Of the two endothelial cell lines immortalized by pmT, and therefore wild-type for p53, the FAK+ line exhibited a slightly higher level of apoptosis upon serum removal (6.3 vs. 2.1%). However, the FAK− line showed a much greater (7.8-fold) increase in apoptosis upon serum withdrawal (24.1 vs. 3.1%).

Figure 1

FAK suppresses a p53-regulated apoptosis pathway in embryoid bodies and endothelial cells. (A) Withdrawal of serum induces apoptosis in FAK− EB generated from wild-type and FAK− TT2 ES cells (Ilić et al., 1995_a_). Cells were cultured for 11 d without leukemia inhibitory factor and in the presence of serum to initiate differentiation. They were then cultured for an additional 24 h either with (+), or without (−) serum. The presence of apoptotic cells was assessed by TUNEL staining, and nuclei were visualized with Hoechst as described in the Materials and Methods. (B) p53 deficiency permits survival, despite the absence of FAK, in anchorage-dependent, serum-deprived endothelial cells. Endothelial cells were isolated from FAK+ or FAK− EB or embryos and immortalized by pmT or incorporation of mutated p53, as described in the Materials and Methods. Apoptosis was assessed by a flow cytometric assay: PI, propidium iodide; Hoechst, Hoechst 33342. In each plot, the events represented in red (upper gated area) correspond to the PI/Hoechst ratios characteristic of an apoptotic population, whereas the events represented in green (lower gated area) correspond to nonapoptotic cells (Hamel et al., 1996). Endothelial cell lines were cultured for 48 h in the presence of serum and for an additional 24 h in the presence (+) or absence (−) of serum. All endothelial lines cultured in the presence of serum, as well as both p53-deficient cell lines cultured without serum, displayed low levels of apoptosis. Of the two endothelial cell lines immortalized by pmT, and therefore wild-type for p53, the FAK+ line exhibited a slightly higher level of apoptosis upon serum removal (6.3 vs. 2.1%). However, the FAK− line showed a much greater (7.8-fold) increase in apoptosis upon serum withdrawal (24.1 vs. 3.1%).

Figure 2

Specific matrices signal cell survival in the absence of serum in both endothelial cells and fibroblasts. Tissue culture plastic wells were either coated with fibronectin (FN), vitronectin (VN), laminin-1 (LA), or type-I collagen (CO), or not coated (NC), as described in the Materials and Methods. Cells were plated for 16 h (primary rabbit synovial fibroblasts: RSF) or 24 h (EpmTp53+FAK+ endothelial cells) on these matrices in the absence of serum. SE, serum control. Apoptotic cells in the culture were quantified as described in Materials and Methods. Each sample of at least 100 cells was obtained in triplicate in each experiment. Experiments were repeated at least three times. Error bars show SD. (A) FAK+p53+ endothelial cells. (B) RSF.

Figure 3

GFP–FAT acts as a dominant negative for FAK. (A) RSF were transfected with cDNA encoding a fusion protein containing GFP and FAK, FRNK, FAT, or FATΔ13C (FAT with deletion of 13 COOH-terminal amino acids). (B) At 8 h, all fusion proteins except GFP– FATΔ13C were detected both in focal contacts and diffusely in the cytoplasm. (C) Quantification of the apoptotic index at 16 h (percentage of transfected [_green_] cells containing condensed, fragmenting nuclei, as detected by Hoechst 33342) for GFP, GFP–FAK, GFP– FAT, GFP–FRNK, and GFP– FATΔ13C transfectants. In contrast to the high apoptotic index (75%) after GFP– FAT transfection, transfections with GFP alone or with GFP fused to intact FAK caused no apoptosis above levels observed in cells plated on fibronectin in the absence of serum (∼20%). GFP–FATΔ13C, which was not detected in focal adhesions, was also unable to induce apoptosis to the same degree as GFP–FAT. (D) Apoptosis assessed by annexin binding and Hoechst 33342 staining. Most RSF expressing GFP–FAT, but not GFP alone or GFP-FAK, are rounded, and their Hoechst 33342-stained nuclei are bright and condensed by 16 h after transfection (arrows). These same cells also show annexin-positive staining (red) in the plasma membrane, indicating they are apoptotic (staining described in Materials and Methods). Untransfected and GFP–FAK–transfected cells are spread (Phase) and have normal nuclei (Hoechst).

Figure 3

GFP–FAT acts as a dominant negative for FAK. (A) RSF were transfected with cDNA encoding a fusion protein containing GFP and FAK, FRNK, FAT, or FATΔ13C (FAT with deletion of 13 COOH-terminal amino acids). (B) At 8 h, all fusion proteins except GFP– FATΔ13C were detected both in focal contacts and diffusely in the cytoplasm. (C) Quantification of the apoptotic index at 16 h (percentage of transfected [_green_] cells containing condensed, fragmenting nuclei, as detected by Hoechst 33342) for GFP, GFP–FAK, GFP– FAT, GFP–FRNK, and GFP– FATΔ13C transfectants. In contrast to the high apoptotic index (75%) after GFP– FAT transfection, transfections with GFP alone or with GFP fused to intact FAK caused no apoptosis above levels observed in cells plated on fibronectin in the absence of serum (∼20%). GFP–FATΔ13C, which was not detected in focal adhesions, was also unable to induce apoptosis to the same degree as GFP–FAT. (D) Apoptosis assessed by annexin binding and Hoechst 33342 staining. Most RSF expressing GFP–FAT, but not GFP alone or GFP-FAK, are rounded, and their Hoechst 33342-stained nuclei are bright and condensed by 16 h after transfection (arrows). These same cells also show annexin-positive staining (red) in the plasma membrane, indicating they are apoptotic (staining described in Materials and Methods). Untransfected and GFP–FAK–transfected cells are spread (Phase) and have normal nuclei (Hoechst).

Figure 3

GFP–FAT acts as a dominant negative for FAK. (A) RSF were transfected with cDNA encoding a fusion protein containing GFP and FAK, FRNK, FAT, or FATΔ13C (FAT with deletion of 13 COOH-terminal amino acids). (B) At 8 h, all fusion proteins except GFP– FATΔ13C were detected both in focal contacts and diffusely in the cytoplasm. (C) Quantification of the apoptotic index at 16 h (percentage of transfected [_green_] cells containing condensed, fragmenting nuclei, as detected by Hoechst 33342) for GFP, GFP–FAK, GFP– FAT, GFP–FRNK, and GFP– FATΔ13C transfectants. In contrast to the high apoptotic index (75%) after GFP– FAT transfection, transfections with GFP alone or with GFP fused to intact FAK caused no apoptosis above levels observed in cells plated on fibronectin in the absence of serum (∼20%). GFP–FATΔ13C, which was not detected in focal adhesions, was also unable to induce apoptosis to the same degree as GFP–FAT. (D) Apoptosis assessed by annexin binding and Hoechst 33342 staining. Most RSF expressing GFP–FAT, but not GFP alone or GFP-FAK, are rounded, and their Hoechst 33342-stained nuclei are bright and condensed by 16 h after transfection (arrows). These same cells also show annexin-positive staining (red) in the plasma membrane, indicating they are apoptotic (staining described in Materials and Methods). Untransfected and GFP–FAK–transfected cells are spread (Phase) and have normal nuclei (Hoechst).

Figure 3

GFP–FAT acts as a dominant negative for FAK. (A) RSF were transfected with cDNA encoding a fusion protein containing GFP and FAK, FRNK, FAT, or FATΔ13C (FAT with deletion of 13 COOH-terminal amino acids). (B) At 8 h, all fusion proteins except GFP– FATΔ13C were detected both in focal contacts and diffusely in the cytoplasm. (C) Quantification of the apoptotic index at 16 h (percentage of transfected [_green_] cells containing condensed, fragmenting nuclei, as detected by Hoechst 33342) for GFP, GFP–FAK, GFP– FAT, GFP–FRNK, and GFP– FATΔ13C transfectants. In contrast to the high apoptotic index (75%) after GFP– FAT transfection, transfections with GFP alone or with GFP fused to intact FAK caused no apoptosis above levels observed in cells plated on fibronectin in the absence of serum (∼20%). GFP–FATΔ13C, which was not detected in focal adhesions, was also unable to induce apoptosis to the same degree as GFP–FAT. (D) Apoptosis assessed by annexin binding and Hoechst 33342 staining. Most RSF expressing GFP–FAT, but not GFP alone or GFP-FAK, are rounded, and their Hoechst 33342-stained nuclei are bright and condensed by 16 h after transfection (arrows). These same cells also show annexin-positive staining (red) in the plasma membrane, indicating they are apoptotic (staining described in Materials and Methods). Untransfected and GFP–FAK–transfected cells are spread (Phase) and have normal nuclei (Hoechst).

Figure 4

Assessment of GFP–FRNK and GFP–FAT function. (A) Cells expressing GFP–FRNK show delayed spreading. RSF transfected with GFP, GFP–FRNK, or GFP–FAT for 10 h were trypsinized and replated on fibronectin-coated coverslips. 60 min after replating, GFP-transfected cells were completely spread, whereas GFP–FRNK and GFP–FAT transfectants were still rounded. By 2 h, GFP–FRNK–transfected cells had spread, showing focal contact localization of the fusion protein (arrowheads). However, a majority of GFP–FAT–transfected cells remained round, although still attached. (B) Transfected cells express similar amounts of GFP–FRNK and GFP–FAT. Cells expressing GFP–FRNK or GFP–FAT were sorted by FACS® 10 h after transfection. An equal number of sorted cells (5 × 103) of each type were lysed in RIPA buffer, separated on 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-GFP antibody (Zymed Laboratories, So. San Francisco, CA). Bands were visualized by ECL-Plus detection system (Amersham Corp., Arlington Heights, IL).

Figure 4

Assessment of GFP–FRNK and GFP–FAT function. (A) Cells expressing GFP–FRNK show delayed spreading. RSF transfected with GFP, GFP–FRNK, or GFP–FAT for 10 h were trypsinized and replated on fibronectin-coated coverslips. 60 min after replating, GFP-transfected cells were completely spread, whereas GFP–FRNK and GFP–FAT transfectants were still rounded. By 2 h, GFP–FRNK–transfected cells had spread, showing focal contact localization of the fusion protein (arrowheads). However, a majority of GFP–FAT–transfected cells remained round, although still attached. (B) Transfected cells express similar amounts of GFP–FRNK and GFP–FAT. Cells expressing GFP–FRNK or GFP–FAT were sorted by FACS® 10 h after transfection. An equal number of sorted cells (5 × 103) of each type were lysed in RIPA buffer, separated on 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-GFP antibody (Zymed Laboratories, So. San Francisco, CA). Bands were visualized by ECL-Plus detection system (Amersham Corp., Arlington Heights, IL).

Figure 5

The PI3K/AKT pathway cannot rescue the apoptotic response of RSF to GFP–FAT. In RSF cultured on fibronectin without serum, blockade of PI3K activity by LY294002 did not trigger apoptosis, nor did cotransfection of constitutively activated AKT (myr AKT) along with GFP–FAT block apoptosis. This suggests that PI3K/AKT are not downstream mediators of the FAK survival pathway in serum-deprived, anchorage-dependent cells.

Figure 6

Characterization of the apoptotic pathway triggered in the absence of ECM survival signals in serum-deprived RSF: pharmacological and dominant-negative strategies reveal an apoptotic pathway that is regulated by p53, activated by cPLA2 and PKC λ/ι, and resistant to CrmA. (A) Blocking the function of p53 permits survival of primary RSF despite suppression of FAK function by GFP–FAT. GFP–FAT was cotransfected into fibroblasts plated on fibronectin, with or without either E1B 19K, Bcl2, or a GSE corresponding to the COOH-terminal domain of p53 (C-term p53), which inactivates p53 function (see Materials and Methods). The apoptotic index was high in GFP–FAT–transfected cells, but greatly reduced in cells cotransfected with GFP–FAT and one of the following: E1B 19K, Bcl2, or C-term p53. However, cotransfection of C-term p53, which contained mutated putative PKC phosphorylation sites (mut C-term p53), with GFP–FAT did not rescue cells from apoptosis. (B–D) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires PKC λ/ι. (B) Inhibitors that block function of all isoforms of PKC (bisindolylmaleimide I and chelerythrine chloride) suppressed GFP–FAT–induced apoptosis, whereas calphostin C, which blocks only the PMA-sensitive isoforms (and therefore not PKC λ/ι), did not. (C) RSF express three isotypes of PKC. To detect PKC isotypes, cells were lysed using RIPA buffer in the presence of proteinase inhibitors. PKCs were immunoprecipitated and detected with anti-PKC α, β, γ, δ, ε, ζ, o, ι, λ, and μ antibodies (Transduction Laboratories, Lexington, KY) after separation in 10% SDS–polyacrylamide gels. Only PKC ε, λ, and ι were detected. + control, PKC isoform standard; − control, precipitation of RSF lysate with nonimmune IgG; anti-PKC, precipitation with antibodies specific for the PKC isoforms. (D). Coexpression of dominant-negative (DN) PKC λ/ι, but not DN PKC ε, blocked GFP–FAT–triggered apoptosis. Cotransfection of the wild-type PKC isoforms with GFP–FAT did not rescue cells from apoptosis. Transfection of wild-type isoforms into GFP-transfected cells did not promote apoptosis. (E) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires cPLA2. Arachidonic acid induced apoptosis in nontransfected or GFP-transfected RSF. Phospholipases catalyze the release of arachidonic acid from phospholipids. AACOCF3, an inhibitor of cPLA2, but not inhibitors of secretory and Ca2+-independent PLA2 or inhibitors of PLC, rescued cells from apoptosis triggered by GFP–FAT. The panPKC inhibitor bisindolylmaleimide I blocked apoptosis triggered by arachidonic acid, suggesting that cPLA2 is upstream of PKC λ/ι in this pathway. (F) Effects of blocking large and small prodomain caspase functions on apoptosis in GFP–FAT–transfected primary RSF. RSF were cotransfected with GFP–FAT and CrmA or with GFP–FAT alone. At 16 h, the apoptotic index of RSF transfected with GFP–FAT was high whether or not CrmA was also transfected. The small prodomain caspase inhibitor, Z-VAD-FMK, inhibited formation of condensed and fragmented nuclei in serum-deprived GFP–FAT–transfected fibroblasts plated on fibronectin.

Figure 6

Characterization of the apoptotic pathway triggered in the absence of ECM survival signals in serum-deprived RSF: pharmacological and dominant-negative strategies reveal an apoptotic pathway that is regulated by p53, activated by cPLA2 and PKC λ/ι, and resistant to CrmA. (A) Blocking the function of p53 permits survival of primary RSF despite suppression of FAK function by GFP–FAT. GFP–FAT was cotransfected into fibroblasts plated on fibronectin, with or without either E1B 19K, Bcl2, or a GSE corresponding to the COOH-terminal domain of p53 (C-term p53), which inactivates p53 function (see Materials and Methods). The apoptotic index was high in GFP–FAT–transfected cells, but greatly reduced in cells cotransfected with GFP–FAT and one of the following: E1B 19K, Bcl2, or C-term p53. However, cotransfection of C-term p53, which contained mutated putative PKC phosphorylation sites (mut C-term p53), with GFP–FAT did not rescue cells from apoptosis. (B–D) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires PKC λ/ι. (B) Inhibitors that block function of all isoforms of PKC (bisindolylmaleimide I and chelerythrine chloride) suppressed GFP–FAT–induced apoptosis, whereas calphostin C, which blocks only the PMA-sensitive isoforms (and therefore not PKC λ/ι), did not. (C) RSF express three isotypes of PKC. To detect PKC isotypes, cells were lysed using RIPA buffer in the presence of proteinase inhibitors. PKCs were immunoprecipitated and detected with anti-PKC α, β, γ, δ, ε, ζ, o, ι, λ, and μ antibodies (Transduction Laboratories, Lexington, KY) after separation in 10% SDS–polyacrylamide gels. Only PKC ε, λ, and ι were detected. + control, PKC isoform standard; − control, precipitation of RSF lysate with nonimmune IgG; anti-PKC, precipitation with antibodies specific for the PKC isoforms. (D). Coexpression of dominant-negative (DN) PKC λ/ι, but not DN PKC ε, blocked GFP–FAT–triggered apoptosis. Cotransfection of the wild-type PKC isoforms with GFP–FAT did not rescue cells from apoptosis. Transfection of wild-type isoforms into GFP-transfected cells did not promote apoptosis. (E) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires cPLA2. Arachidonic acid induced apoptosis in nontransfected or GFP-transfected RSF. Phospholipases catalyze the release of arachidonic acid from phospholipids. AACOCF3, an inhibitor of cPLA2, but not inhibitors of secretory and Ca2+-independent PLA2 or inhibitors of PLC, rescued cells from apoptosis triggered by GFP–FAT. The panPKC inhibitor bisindolylmaleimide I blocked apoptosis triggered by arachidonic acid, suggesting that cPLA2 is upstream of PKC λ/ι in this pathway. (F) Effects of blocking large and small prodomain caspase functions on apoptosis in GFP–FAT–transfected primary RSF. RSF were cotransfected with GFP–FAT and CrmA or with GFP–FAT alone. At 16 h, the apoptotic index of RSF transfected with GFP–FAT was high whether or not CrmA was also transfected. The small prodomain caspase inhibitor, Z-VAD-FMK, inhibited formation of condensed and fragmented nuclei in serum-deprived GFP–FAT–transfected fibroblasts plated on fibronectin.

Figure 6

Characterization of the apoptotic pathway triggered in the absence of ECM survival signals in serum-deprived RSF: pharmacological and dominant-negative strategies reveal an apoptotic pathway that is regulated by p53, activated by cPLA2 and PKC λ/ι, and resistant to CrmA. (A) Blocking the function of p53 permits survival of primary RSF despite suppression of FAK function by GFP–FAT. GFP–FAT was cotransfected into fibroblasts plated on fibronectin, with or without either E1B 19K, Bcl2, or a GSE corresponding to the COOH-terminal domain of p53 (C-term p53), which inactivates p53 function (see Materials and Methods). The apoptotic index was high in GFP–FAT–transfected cells, but greatly reduced in cells cotransfected with GFP–FAT and one of the following: E1B 19K, Bcl2, or C-term p53. However, cotransfection of C-term p53, which contained mutated putative PKC phosphorylation sites (mut C-term p53), with GFP–FAT did not rescue cells from apoptosis. (B–D) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires PKC λ/ι. (B) Inhibitors that block function of all isoforms of PKC (bisindolylmaleimide I and chelerythrine chloride) suppressed GFP–FAT–induced apoptosis, whereas calphostin C, which blocks only the PMA-sensitive isoforms (and therefore not PKC λ/ι), did not. (C) RSF express three isotypes of PKC. To detect PKC isotypes, cells were lysed using RIPA buffer in the presence of proteinase inhibitors. PKCs were immunoprecipitated and detected with anti-PKC α, β, γ, δ, ε, ζ, o, ι, λ, and μ antibodies (Transduction Laboratories, Lexington, KY) after separation in 10% SDS–polyacrylamide gels. Only PKC ε, λ, and ι were detected. + control, PKC isoform standard; − control, precipitation of RSF lysate with nonimmune IgG; anti-PKC, precipitation with antibodies specific for the PKC isoforms. (D). Coexpression of dominant-negative (DN) PKC λ/ι, but not DN PKC ε, blocked GFP–FAT–triggered apoptosis. Cotransfection of the wild-type PKC isoforms with GFP–FAT did not rescue cells from apoptosis. Transfection of wild-type isoforms into GFP-transfected cells did not promote apoptosis. (E) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires cPLA2. Arachidonic acid induced apoptosis in nontransfected or GFP-transfected RSF. Phospholipases catalyze the release of arachidonic acid from phospholipids. AACOCF3, an inhibitor of cPLA2, but not inhibitors of secretory and Ca2+-independent PLA2 or inhibitors of PLC, rescued cells from apoptosis triggered by GFP–FAT. The panPKC inhibitor bisindolylmaleimide I blocked apoptosis triggered by arachidonic acid, suggesting that cPLA2 is upstream of PKC λ/ι in this pathway. (F) Effects of blocking large and small prodomain caspase functions on apoptosis in GFP–FAT–transfected primary RSF. RSF were cotransfected with GFP–FAT and CrmA or with GFP–FAT alone. At 16 h, the apoptotic index of RSF transfected with GFP–FAT was high whether or not CrmA was also transfected. The small prodomain caspase inhibitor, Z-VAD-FMK, inhibited formation of condensed and fragmented nuclei in serum-deprived GFP–FAT–transfected fibroblasts plated on fibronectin.

Figure 6

Characterization of the apoptotic pathway triggered in the absence of ECM survival signals in serum-deprived RSF: pharmacological and dominant-negative strategies reveal an apoptotic pathway that is regulated by p53, activated by cPLA2 and PKC λ/ι, and resistant to CrmA. (A) Blocking the function of p53 permits survival of primary RSF despite suppression of FAK function by GFP–FAT. GFP–FAT was cotransfected into fibroblasts plated on fibronectin, with or without either E1B 19K, Bcl2, or a GSE corresponding to the COOH-terminal domain of p53 (C-term p53), which inactivates p53 function (see Materials and Methods). The apoptotic index was high in GFP–FAT–transfected cells, but greatly reduced in cells cotransfected with GFP–FAT and one of the following: E1B 19K, Bcl2, or C-term p53. However, cotransfection of C-term p53, which contained mutated putative PKC phosphorylation sites (mut C-term p53), with GFP–FAT did not rescue cells from apoptosis. (B–D) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires PKC λ/ι. (B) Inhibitors that block function of all isoforms of PKC (bisindolylmaleimide I and chelerythrine chloride) suppressed GFP–FAT–induced apoptosis, whereas calphostin C, which blocks only the PMA-sensitive isoforms (and therefore not PKC λ/ι), did not. (C) RSF express three isotypes of PKC. To detect PKC isotypes, cells were lysed using RIPA buffer in the presence of proteinase inhibitors. PKCs were immunoprecipitated and detected with anti-PKC α, β, γ, δ, ε, ζ, o, ι, λ, and μ antibodies (Transduction Laboratories, Lexington, KY) after separation in 10% SDS–polyacrylamide gels. Only PKC ε, λ, and ι were detected. + control, PKC isoform standard; − control, precipitation of RSF lysate with nonimmune IgG; anti-PKC, precipitation with antibodies specific for the PKC isoforms. (D). Coexpression of dominant-negative (DN) PKC λ/ι, but not DN PKC ε, blocked GFP–FAT–triggered apoptosis. Cotransfection of the wild-type PKC isoforms with GFP–FAT did not rescue cells from apoptosis. Transfection of wild-type isoforms into GFP-transfected cells did not promote apoptosis. (E) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires cPLA2. Arachidonic acid induced apoptosis in nontransfected or GFP-transfected RSF. Phospholipases catalyze the release of arachidonic acid from phospholipids. AACOCF3, an inhibitor of cPLA2, but not inhibitors of secretory and Ca2+-independent PLA2 or inhibitors of PLC, rescued cells from apoptosis triggered by GFP–FAT. The panPKC inhibitor bisindolylmaleimide I blocked apoptosis triggered by arachidonic acid, suggesting that cPLA2 is upstream of PKC λ/ι in this pathway. (F) Effects of blocking large and small prodomain caspase functions on apoptosis in GFP–FAT–transfected primary RSF. RSF were cotransfected with GFP–FAT and CrmA or with GFP–FAT alone. At 16 h, the apoptotic index of RSF transfected with GFP–FAT was high whether or not CrmA was also transfected. The small prodomain caspase inhibitor, Z-VAD-FMK, inhibited formation of condensed and fragmented nuclei in serum-deprived GFP–FAT–transfected fibroblasts plated on fibronectin.

Figure 6

Characterization of the apoptotic pathway triggered in the absence of ECM survival signals in serum-deprived RSF: pharmacological and dominant-negative strategies reveal an apoptotic pathway that is regulated by p53, activated by cPLA2 and PKC λ/ι, and resistant to CrmA. (A) Blocking the function of p53 permits survival of primary RSF despite suppression of FAK function by GFP–FAT. GFP–FAT was cotransfected into fibroblasts plated on fibronectin, with or without either E1B 19K, Bcl2, or a GSE corresponding to the COOH-terminal domain of p53 (C-term p53), which inactivates p53 function (see Materials and Methods). The apoptotic index was high in GFP–FAT–transfected cells, but greatly reduced in cells cotransfected with GFP–FAT and one of the following: E1B 19K, Bcl2, or C-term p53. However, cotransfection of C-term p53, which contained mutated putative PKC phosphorylation sites (mut C-term p53), with GFP–FAT did not rescue cells from apoptosis. (B–D) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires PKC λ/ι. (B) Inhibitors that block function of all isoforms of PKC (bisindolylmaleimide I and chelerythrine chloride) suppressed GFP–FAT–induced apoptosis, whereas calphostin C, which blocks only the PMA-sensitive isoforms (and therefore not PKC λ/ι), did not. (C) RSF express three isotypes of PKC. To detect PKC isotypes, cells were lysed using RIPA buffer in the presence of proteinase inhibitors. PKCs were immunoprecipitated and detected with anti-PKC α, β, γ, δ, ε, ζ, o, ι, λ, and μ antibodies (Transduction Laboratories, Lexington, KY) after separation in 10% SDS–polyacrylamide gels. Only PKC ε, λ, and ι were detected. + control, PKC isoform standard; − control, precipitation of RSF lysate with nonimmune IgG; anti-PKC, precipitation with antibodies specific for the PKC isoforms. (D). Coexpression of dominant-negative (DN) PKC λ/ι, but not DN PKC ε, blocked GFP–FAT–triggered apoptosis. Cotransfection of the wild-type PKC isoforms with GFP–FAT did not rescue cells from apoptosis. Transfection of wild-type isoforms into GFP-transfected cells did not promote apoptosis. (E) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires cPLA2. Arachidonic acid induced apoptosis in nontransfected or GFP-transfected RSF. Phospholipases catalyze the release of arachidonic acid from phospholipids. AACOCF3, an inhibitor of cPLA2, but not inhibitors of secretory and Ca2+-independent PLA2 or inhibitors of PLC, rescued cells from apoptosis triggered by GFP–FAT. The panPKC inhibitor bisindolylmaleimide I blocked apoptosis triggered by arachidonic acid, suggesting that cPLA2 is upstream of PKC λ/ι in this pathway. (F) Effects of blocking large and small prodomain caspase functions on apoptosis in GFP–FAT–transfected primary RSF. RSF were cotransfected with GFP–FAT and CrmA or with GFP–FAT alone. At 16 h, the apoptotic index of RSF transfected with GFP–FAT was high whether or not CrmA was also transfected. The small prodomain caspase inhibitor, Z-VAD-FMK, inhibited formation of condensed and fragmented nuclei in serum-deprived GFP–FAT–transfected fibroblasts plated on fibronectin.

Figure 6

Characterization of the apoptotic pathway triggered in the absence of ECM survival signals in serum-deprived RSF: pharmacological and dominant-negative strategies reveal an apoptotic pathway that is regulated by p53, activated by cPLA2 and PKC λ/ι, and resistant to CrmA. (A) Blocking the function of p53 permits survival of primary RSF despite suppression of FAK function by GFP–FAT. GFP–FAT was cotransfected into fibroblasts plated on fibronectin, with or without either E1B 19K, Bcl2, or a GSE corresponding to the COOH-terminal domain of p53 (C-term p53), which inactivates p53 function (see Materials and Methods). The apoptotic index was high in GFP–FAT–transfected cells, but greatly reduced in cells cotransfected with GFP–FAT and one of the following: E1B 19K, Bcl2, or C-term p53. However, cotransfection of C-term p53, which contained mutated putative PKC phosphorylation sites (mut C-term p53), with GFP–FAT did not rescue cells from apoptosis. (B–D) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires PKC λ/ι. (B) Inhibitors that block function of all isoforms of PKC (bisindolylmaleimide I and chelerythrine chloride) suppressed GFP–FAT–induced apoptosis, whereas calphostin C, which blocks only the PMA-sensitive isoforms (and therefore not PKC λ/ι), did not. (C) RSF express three isotypes of PKC. To detect PKC isotypes, cells were lysed using RIPA buffer in the presence of proteinase inhibitors. PKCs were immunoprecipitated and detected with anti-PKC α, β, γ, δ, ε, ζ, o, ι, λ, and μ antibodies (Transduction Laboratories, Lexington, KY) after separation in 10% SDS–polyacrylamide gels. Only PKC ε, λ, and ι were detected. + control, PKC isoform standard; − control, precipitation of RSF lysate with nonimmune IgG; anti-PKC, precipitation with antibodies specific for the PKC isoforms. (D). Coexpression of dominant-negative (DN) PKC λ/ι, but not DN PKC ε, blocked GFP–FAT–triggered apoptosis. Cotransfection of the wild-type PKC isoforms with GFP–FAT did not rescue cells from apoptosis. Transfection of wild-type isoforms into GFP-transfected cells did not promote apoptosis. (E) Apoptosis triggered by inactivation of FAK function in serum-deprived anchorage-dependent RSF requires cPLA2. Arachidonic acid induced apoptosis in nontransfected or GFP-transfected RSF. Phospholipases catalyze the release of arachidonic acid from phospholipids. AACOCF3, an inhibitor of cPLA2, but not inhibitors of secretory and Ca2+-independent PLA2 or inhibitors of PLC, rescued cells from apoptosis triggered by GFP–FAT. The panPKC inhibitor bisindolylmaleimide I blocked apoptosis triggered by arachidonic acid, suggesting that cPLA2 is upstream of PKC λ/ι in this pathway. (F) Effects of blocking large and small prodomain caspase functions on apoptosis in GFP–FAT–transfected primary RSF. RSF were cotransfected with GFP–FAT and CrmA or with GFP–FAT alone. At 16 h, the apoptotic index of RSF transfected with GFP–FAT was high whether or not CrmA was also transfected. The small prodomain caspase inhibitor, Z-VAD-FMK, inhibited formation of condensed and fragmented nuclei in serum-deprived GFP–FAT–transfected fibroblasts plated on fibronectin.

Figure 7

Model for the transmission of matrix survival signals. Signals from fibronectin are detected by fibronectin receptors and transduced by FAK. These signals suppress a p53-dependent apoptotic pathway in anchorage-dependent, serum-deprived fibroblasts. When both ECM and serum survival signals are absent, PKC λ/ι is strongly implicated in the activation of p53, while pharmacological evidence also supports a role for cPLA2. FADD, Fas-associated death domain– containing protein; LPDC, large prodomain caspases; SPDC, small prodomain caspases; TRADD, TNF receptor 1–associated death domain protein.

References

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