Human astrocytes are resistant to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis

Abstract

Human astrocytes express Fas yet are resistant to Fas-induced apoptosis. Here, we report that calcium/calmodulin-dependent protein kinase II (CaMKII) is constitutively activated in human astrocytes and protects the cells from apoptotic stimulation by Fas agonist. Once stimulated, Fas recruits Fas-associated death domain and caspase-8 for the assembly of the death-inducing signaling complex (DISC); however, caspase-8 cleavage is inhibited in the DISC. Inhibition of CaMKII kinase activity inhibits the expression of phosphoprotein enriched astrocytes-15 kDa/phosphoprotein enriched in diabetes (PEA-15/PED) and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP), thus releasing their inhibition of caspase-8 cleavage. Inhibition of PEA-15/PED or c-FLIP by small interfering RNA sensitizes human astrocytes to Fas-induced apoptosis. In contrast, inhibition of CaMKII, PEA-15, or c-FLIP does not affect the sensitivity of human astrocytes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL death receptors (DR4, DR5) are weakly expressed at mRNA, protein, and cell surface levels and thus fail to mediate the assembly of the DISC in human astrocytes. Overexpression of DR5 restores TRAIL signaling pathways and sensitizes the human astrocytes to TRAIL-induced apoptosis if CaMKII kinase activity or expression of PEA-15 and c-FLIP is inhibited; the results suggest that CaMKII-mediated pathways prevent TRAIL-induced apoptosis in human astrocytes under conditions in which TRAIL death receptors are upregulated. This study has therefore identified the molecular mechanisms that protect normal human astrocytes from apoptosis induced by Fas ligand and TRAIL.

Figure 1.

Human astrocytes are resistant to Fas agonist and TRAIL. A, Flow cytometry analysis of the cell surface expression of Fas, DR4, and DR5 in human astrocytes and A549 and U343MG cancer cell lines. B, Western blot detection of DR4 and DR5 expression in human astrocytes, A549, U343MG, and Cos-7 cells transfected with either DR4 or DR5 cDNA. Three antibodies against DR4 and three antibodies to DR5 used in the study are indicated to the left and are described in Materials and Methods. Extracellular signal-regulated kinase 1/2 (ERK1/2) was used as protein loading control. C, RT-PCR analysis of DR4 and DR5 mRNA expression in human astrocytes and control A549 cells. D, Cell viability in human glioma cell line U343MG treated with the various doses of three recombinant forms of human TRAIL, as described in Materials and Methods. E, Cell viability analysis of human astrocytes after exposure to 3 μg/ml Fas agonist monoclonal antibody (Fas mAb), nontagged TRAIL (TRAIL), histidine-tagged TRAIL (his-TRAIL), and antibody cross-linked Flag-tagged TRAIL (Flag-TRAIL) for 24 h. F, Phase-contrast microscopic observation of apoptotic cell death in human glioma cell line U343MG but not human astrocytes after exposure to 3 μg/ml Fas agonist and various forms of recombinant TRAIL. Cell viability data (D, E) were representative from three independent experiments (mean ± SEM; n = 6).

Figure 2.

Inhibition of CaMKII sensitizes human astrocytes to Fas-induced apoptosis in human astrocytes. A, Western blot analysis of the expression of CaMKII and phosphorylated CaMKII (p-CaMKII, Thr286) in human astrocytes (A) and neurons (N) untreated or treated with KN93 (100 μm) for 24 h. Actin was used as protein loading control. B, CaMKII kinase analysis for the kinase activity in human astrocytes treated with the various doses of KN93 for 24 h (mean ± SEM; n = 3; *p < 0.05). C, Phase-contrast microscopy examination of human astrocytes untreated or treated with TRAIL (1 μg/ml) and Fas agonist monoclonal antibody (Fas mAb; 1 μg/ml) in the absence or presence of KN93 (100 μm) for 24 h. D, Cell viability analysis of human astrocytes after treatment for 24 h with various doses of KN93 in the absence or presence of Fas agonist (1 μg/ml) and TRAIL (1 μg/ml) (mean ± SEM; n = 6). E, Western blot and subcellular fractionation analysis of cleavage of caspases and mitochondrial release of cytochrome c. Human astrocytes were treated with KN93 (100 μm) for 16 h and then with Fas agonist (1 μg/ml) and TRAIL (1 μg/ml) for the times indicated at the top. Human astrocytes were also untreated (Medium) or treated alone with KN93 (100 μm) for 22 h (KN93) as controls. The untreated and treated cells were subject to Western blot analysis of cleavage of caspases and DFF45 (panels 1–4) with actin as the protein loading control (panel 5). Subcellular fractionation of the cells showed the presence of cytochrome c in mitochondria (M.) and its release into cytosol (C.) after the treatment. The antibodies are indicated to the left, and the proteins detected are indicated to the right (A, E).

Figure 3.

Insufficient expression of TRAIL receptors blocks TRAIL-induced signal pathway in human astrocytes. A, B, Analysis of Fas agonist (A) and TRAIL-induced DISC (B) in human astrocytes and human glioma U343MG cells. The DISC was immunoprecipitated by the treatment of human astrocytes with either Fas agonist antibody or Flag-tagged TRAIL for 15 min in stimulated cells (+) or after cell lysis in unstimulated cells (−). Cell lysates obtained from untreated cells (Lysate) were included as control for the expression of the endogenous proteins. C, TRAIL sensitivity of human astrocytes transfected with DR5 cDNA. Human astrocytes were transfected with DR5 cDNA for 48 h and treated with KN93 (100 μm) and TRAIL (1 μg/ml), alone or in combination as indicated at the top. After 6 h of treatment, the cells were subjected to Western blot for DR5 expression (top) and caspase-3 cleavage (middle). After 24 h of treatment, the cells were analyzed for cell viability (bottom) (mean ± SEM; n = 6; *p < 0.05).

Figure 4.

CaMKII inhibitor KN93 inhibits PEA-15/PED and c-FLIP expression. Human astrocytes were treated or untreated (Medium) with KN93 (100 μm) for 24 h and then subjected to flow cytometry analysis of Fas and DR5 expression (A), Western blot analysis of the expression of key apoptotic proteins (B), and two-dimensional PAGE analysis of PEA-15/PED isoforms (C). The antibodies are indicated to the left (B). The isoelectric direction (H⁺, OH⁻) of two-dimensional PAGE is indicated above (C).

Figure 5.

CaMKII dominant negative sensitizes human astrocytes to Fas but not TRAIL. Human astrocytes were transfected with pCD derivative SRα vector expressing α-CaMKII K42M mutant or empty SRα vector for 48 h. Some of the astrocytes transfected with empty SRα vector were untreated or treated with KN93 (100 μm) and Fas agonist (1 μg/ml), alone or in combination, for 6 h for Western blot analysis (A, B) and 24 h for cell viability analysis (C). Some of the astrocytes transfected with the α-CaMKII K42M mutant were untreated or treated with Fas agonist (1 μg/ml) or nontagged TRAIL (1 μg/ml) for 6 h for Western blot analysis (A, B) and 24 h for cell viability analysis (C) (mean ± SEM; n = 6). Human astrocytes untreated (Medium) or treated with Fas agonist (1 μg/ml) were included in the examination as controls. The treatments are indicated at the top, the antibodies used are indicated to the left, and the proteins detected are indicated to the right (A, B). *p < 0.05.

Figure 6.

Effects of siRNA specific to PEA-15/PED and c-FLIP on Fas and TRAIL of human astrocytes. A, Western blot analysis of the expression of PEA-15/PED and c-FLIP (c-FLIP_L, c-FLIP_S). Human astrocytes were transfected with PEA-15/PED and c-FLIP siRNA or nonspecific control siRNA (Mock) for 24 h before being subjected to Western blot. ERK1/2 was used as the protein loading control. B, C, PEA-15/PED or c-FLIP siRNA sensitizes human astrocytes to Fas agonist. The astrocytes transfected with siRNA were treated with Fas agonist antibody (1 μg/ml) for 6 h and examined on Western blots (B) and 24 h for cell viability assay (C). D, E, Human astrocytes were nontransfected (first column) or transfected with DR5 cDNA (the second to 10th columns) for 48 h and then either transfected or nontransfected with PEA-15/PED or c-FLIP siRNA for 24 h. The cells were further treated or untreated with nontagged TRAIL (1 μg/ml) for 6 h for Western blot detection of caspase-8 cleavage (D) or for 24 h for cell viability analysis (E) in which each value represents six samples, and * indicates the significance (p < 0.05) (C).