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First published online 31 July 2007
doi: 10.1242/jcs.03473

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GSK-3 acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 in ceramide-induced mitochondrial apoptosis

¹ Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan 701, Taiwan
² Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan 701, Taiwan
³ Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan 701, Taiwan
⁴ Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan 701, Taiwan
⁵ Department of Microbiology and Immunology, Chung-Shan Medical University, Taichung 402, Taiwan
⁶ Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University Medical College, Tainan 701, Taiwan

View larger version (69K): [in this window] [in a new window]   Fig. 1. Ceramide activates GSK-3. 10I cells were treated with 25 µM of C2-ceramide for 1, 2, 4 and 6 hours with or without 10 mM LiCl. We used western blotting to determine GSK-3 phosphorylation (P-GSK-3) at serine 9 and glycogen synthase phosphorylation (P-GS) at serine 641. Total GSK-3 and GS proteins were the control. The relative ratio of P-GSK-3:total GSK-3 and of hyperphosphorylated GS (hyper P-GS):total GS are shown. -actin levels were determined as an internal control.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Inhibition of GSK-3 blocks ceramide-induced apoptosis. (A) 10I cells were treated with 25 µM of C2-ceramide for 6 hours with or without the GSK-3 inhibitors LiCl (10 mM) and SB216763 (10 µM). Cell apoptosis was determined using DAPI staining and microscopy (to analyze nuclear fragmentation), and propidium iodide (PI) staining and flow cytometric analysis (to analyze DNA fragmentation). Arrowheads indicate apoptotic cells; percentages of apoptotic cells in the subG0 phase are marked in the histograms. (B) 10I cells were treated with 25 µM of C2-ceramide for 6 hours with or without various doses of LiCl and SB216763 as indicated. Cell apoptosis was detected using PI, annexin V and TUNEL staining, followed by flow cytometric analysis. The percentage of apoptotic cells is shown (mean ± s.d. of five replicates for PI staining and of triplicates for annexin V and TUNEL staining). Cells treated with DMSO were used as the reagent control. (C) 10I cells were transfected with 100 nM of GSK-3 siRNA (95.8% transfection efficiency) or 100 nM of GSK-3 siRNA (96.1% transfection efficiency) for 24 hours as described in Materials and Methods. After further treatment with 25 µM of C2-ceramide for 6 hours, the GSK-3, GSK-3, GS phosphorylated at serine 641 (P-GS), and levels of total GS (GS) were detected using western blotting. -actin levels were determined as an internal control. (D) Cells pre-transfected for 24 hours with control siRNA or GSK-3 siRNA (100 nM) were treated with 25 µM C2-ceramide for 6 hours. We used DAPI staining plus microscopy to determine cell apoptosis characterized by nuclear fragmentation, and PI staining plus flow cytometry to determine cell apoptosis characterized by DNA fragmentation. Arrowheads indicate the apoptotic cells; percentages of apoptotic cells in the subG0 phase are given in each micrograph.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Inhibition of GSK-3 blocked ceramide-induced mitochondrial apoptosis. 10I cells were treated with 25 µM of C2-ceramide for 6 hours with or without the GSK-3 inhibitors LiCl (10 and 20 mM) and SB216763 (10 and 20 µM), or pre-transfected for 24 hours with control siRNA (100 nM) or GSK-3 siRNA (50 and 100 nM). (A) We used Rhodamine 123 staining plus flow cytometry to detect the m reduction. Partial histograms are shown. The percentages of cells with m reduction are given as the average of triplicate cultures (mean ± s.d.). (B) We used western blotting to determine the cytosolic levels of cytochrome c. -actin levels were determined as an internal control. (C) We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Leu-Glu(-OMe)-His-Asp(-OMe)-pNA and benzyloxycarbonyl-Asp(-OMe)-Glu(-OMe)-Val-Asp(-OMe)-pNA to determine the activities of caspase-9 and caspase-3. Data are given as the average of triplicate cultures (mean ± s.d.). pNA, p-nitroanilide.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Inhibition of GSK-3 blocks ceramide-induced activation of caspase-2 and caspase-8 and expression of tBid. 10I cells were treated with 25 µM C2-ceramide for 6 hours with or without LiCl (10 and 20 mM) and SB216763 (10 and 20 µM), or were pre-transfected for 24 hours with control siRNA (100 nM) or GSK-3 siRNA (50 and 100 nM). (A) We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-pNA and benzyloxycarbonyl-Ile-Glu(-OMe)-Thr-Asp(-OMe)-pNA to determine the activities of caspase-2 and caspase-8. OD, optical density. Data are given as the average of triplicate cultures (mean ± s.d.). (B) We used western blotting with a tBid-specific antibody to detect tBid expression. -actin levels were determined as an internal control.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Ceramide activates PP2A, which then indirectly activates GSK-3. (A) 10I cells were treated with 25 µM C2-ceramide for 6 hours with or without LiCl or OA. We used western blotting to determine phosphorylated GSK-3 (P-GSK-3). Total GSK-3 protein was the control. The relative ratios of P-GSK-3:total GSK-3 protein are shown. -actin levels were determined as an internal control. (B) 10I cells were transfected with PP2A (0.02 and 0.1 µg) for 6 hours and incubated without or with OA for an additional 6 hours. The transfection efficiency determined using FITC-labeled immunoglobulin was 92.7% for the cells transfected with 0.02 µg PP2A and 95.8% for those transfected with 0.1 µg PP2A. We used western blotting to determine phosphorylated GSK-3; the ratio of P-GSK-3:total GSK-3 protein level is shown. -actin levels were determined as an internal control. (C) We used immunostaining plus confocal microscopy to determine phosphorylated GSK-3 (green), and PI staining (red) for nuclear staining. (D) To determine whether PP2A directly affects GSK-3 dephosphorylation, GSK-3 was immunoprecipitated (IP) from untreated cells and then incubated together with PP2A that had been immunoprecipitated from 25 µM ceramide-treated cells for 30 minutes at 30°C with or without 50 nM OA. After the cells had been washed, we used western blotting to determine P-GSK-3; the relative P-GSK-3:IP GSK-3 protein level is shown. The IP PP2A level was determined using western blotting. PP2A activity was determined using substrate dephosphorylation; relative PP2A activity is shown.

View larger version (66K): [in this window] [in a new window]   Fig. 6. GSK-3 activation is mediated through PP2A-regulated inactivation of the PI 3-kinase-Akt pathway. (A) 10I cells were treated with 25 µM of C2-ceramide for 4 hours with or without LiCl, wortmannin (10 nM) or PD98059 (100 µM). We used western blotting to determine GSK-3 phosphorylation (P-GSK-3) at serine 9; the relative ratio of P-GSK-3: total GSK-3 protein level is shown. -actin expression was an internal control. (B) 10I cells were transfected with 50 nM (88.7% transfection efficiency) of Akt siRNA as described in Materials and Methods. After ceramide treatment, levels of P-GSK-3, GSK-3 and Akt were detected using western blotting. The relative ratio of P-GSK-3:GSK-3 protein level is shown. -actin levels were determined as an internal control. (C) 10I cells were treated with 25 µM of C2-ceramide with or without the PP2A inhibitor OA for different time periods as indicated. We used western blotting to determine phosphorylated GSK-3 (P-GSK-3) and Akt phosphorylated at serine 473 (P-Akt); the relative ratios of P-Akt:total Akt and P-GSK-3:GSK-3 are shown, respectively. -actin expression was an internal control. (D) To determine whether PP2A has a direct effect on Akt dephosphorylation, Akt was immunoprecipitated (IP) from untreated cells and then incubated together with PP2A that had been immunoprecipitated from 25 µM of ceramide-treated cells for 30 minutes at 30°C with or without 50 nM of OA. After washing, we used western blotting to determine P-Akt; the relative ratio of P-Akt:IP Akt protein levels is shown.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Schematic diagram of the role of GSK-3 in the signaling pathways of ceramide-induced mitochondrial apoptosis. During ceramide-induced apoptosis, activated PP2A induces GSK-3 dephosphorylation and activation indirectly through a PI 3-kinase-Akt-regulated pathway. How ceramide activates PP2A remains unknown. Also, how GSK-3 activates caspase-2 and caspase-8, which causes mitochondrial damage and apoptosis, needs to be deciphered. It is of particular interest to know whether there is cross-talk between GSK-3 and Bcl-2.

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