Glycogen Synthase Kinase-3 Mediates Endoplasmic Reticulum Stress-Induced Lysosomal Apoptosis in Leukemia

Glycogen synthase kinase (GSK)-3 may modulate endoplasmic reticulum (ER) stress-induced apoptosis; however, the mechanism remains unclear. Our data showed that human monocytic leukemia/lymphoma U937 and acute myeloid leukemia HL-60, but not chronic myeloid leukemia K562, cells were susceptible to apoptosis induced by ER stressor tunicamycin, a protein glycosylation inhibitor. Tunicamycin caused early activation of caspase-2, -3, -4, and -8, followed by apoptosis, whereas caspase-9 was slowly activated. Inhibiting caspase-2 reduced activation of caspase-8 and -3 but had no effect on caspase-4. Tunicamycin induced apoptosis independently of the mitochondrial pathway but caused lysosomal destabilization followed by lysosomal membrane permeabilization (LMP), cathepsin B relocation from lysosomes to the cytosol, and caspase-8 and -3 activation. It is notable that caspase-2 mediated lysosomal destabilization. Inhibiting GSK-3 comprehensively reduced lysosomal apoptosis after caspase-2 inhibition. Unlike U937 and HL-60 cells, K562 cells showed nonresponsive ER stress and failure of activation of GSK-3 and caspase-2 in response to tunicamycin. Activating GSK-3 caused K562 cells to be susceptible to tunicamycin-induced apoptosis. Taken together, we show that GSK-3 exhibits a mechanism of ER stress-induced lysosomal apoptosis in leukemia involving caspase-2-induced LMP and cathepsin B relocation, which result in caspase-8 and -3 activation. Leukemia is pathologically characterized by abnormal expansion of hematopoietic progenitor cells. Several drugs have been developed for therapeutic applications; however, leukemia remains problematic because of relapse and drug resistance. The proapoptotic action of antileukemic agents may act through the induction of endoplasmic reticulum (ER) stress (Du et al., 2006; Rahmani et al., 2007). Therefore, cellular ER is a target for improved, selective anticancer therapies (Linder and Shoshan, 2005; Boelens et al., 2007). However, the molecular mechanism of ER stress-triggered apoptosis in leukemia remains unresolved. The ER is the site of protein synthesis, modification, and folding. Multiple insults, such as inhibition of glycosylation, reduction of disulfide bonds, calcium depletion from the ER lumen, impairment of protein transport to the Golgi, and expression of mutated proteins in the ER, can trigger unfolded protein response (UPR) after ER stress (Wu and Kaufman, 2006). These events enhance protein folding and degradation within the ER and down-regulate protein synthesis until cells have recovered from ER stress. However, prolonged ER stress may eventually cause apoptosis, whereas calcium homeostasis and UPR cannot be restored. In ER stress-induced apoptosis, several apoptotic signaling pathways are demonstrated (Breckenridge et al., 2003). ER stress-induced CCAAT/enhancer-binding protein homoloThis work was supported by the National Science Council, Taiwan [Grant NSC 96-2320-B-006-018-MY3]; and the National Cheng Kung University, Taiwan [Landmark Project C020]. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.148122. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: ER, endoplasmic reticulum; UPR, unfolded protein response; CHOP, CCAAT/enhancer-binding protein homologous protein; LMP, lysosomal membrane permeabilization; GSK, glycogen synthase kinase; Z-FA-fmk, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone; pepstatin A, Isovalery-Val-Val-Sta-Ala-Sta; SB415286, 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; DMSO, dimethyl sulfoxide; DAPI, 4,6-diamidino-2-phenylindole; PI, propidium iodide; AO, acridine orange; Z-VDVAD-fmk, benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-fluoromethyl ketone; Z-IETD-fmk, benzyloxycarbonyl-Ile-Glu(Ome)-Thr-Asp(Ome)-fluoromethyl ketone; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; GRP, glucose-regulated protein; PBS, phosphate-buffered saline; HIAP2, human inhibitor of apoptosis 2; PP2A, protein phosphatase 2A; 17-AAG, 17-(allylamino)17-demethoxygeldanamycin. 0022-3565/09/3292-524–531$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 148122/3457833 JPET 329:524–531, 2009 Printed in U.S.A. 524 http://jpet.aspetjournals.org/content/suppl/2009/02/02/jpet.108.148122.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from gous protein (CHOP/Gadd153), a transcription factor that suppresses the expression of antiapoptotic protein Bcl-2 and increases reactive oxygen species production, is involved in apoptosis through the mitochondrial pathway (McCullough et al., 2001). ER stress-activated c-Jun NH2-terminal kinase is also involved (Tan et al., 2006). Calpain, a calcium-dependent protease, generally causes activation of human caspase-4, a specific ER stress-activated caspase with 48% sequence homology to murine caspase-12 (Nakagawa and Yuan, 2000; Fischer et al., 2002). It is notable that caspase-4 triggers apoptotic pathways dependent or independent of caspase-9 and -3 activation (Breckenridge et al., 2003). Furthermore, activation of caspase-2, -3, -7, -8, and -9 also have been reported in ER stress-induced apoptosis (Dahmer, 2005; Cheung et al., 2006; Liu et al., 2006; Upton et al., 2008). However, the role of caspase cascade activation is still controversial owing to differences in ER stressor stimulation and cell type dependence. The lysosome, an acidic organelle, plays a pivotal role in apoptosis caused by various stimuli, including oxidative stress, tumor necrosis factor, sphingosine, p53, and staurosporine (Ferri and Kroemer, 2001; Guicciardi et al., 2004). However, the precise lysosomal pathway in ER stress-induced apoptosis remains unclear. Apoptotic stimuli cause lysosomal membrane permeabilization (LMP) through a variety of regulatory factors, such as calcium, reactive oxygen species, ceramide, sphingosine, phospholipase, Bax, Bim, Bid, and caspase (Guicciardi et al., 2004; Kroemer and Jaattela, 2005). The lysosomal proteolytic enzymes, mainly proteases of the cathepsin family, translocate to the cytosol. Once in the cytosol, cathepsin B and cathepsin D are the major mediators triggering apoptotic pathways, which involve Bid truncation, caspase activation, and mitochondrial damage (Chwieralski et al., 2006). Under ER stress, both lysosomal and mitochondrial destabilization may contribute to the initiation stage of apoptosis. Glycogen synthase kinase (GSK)-3 , a serine/threonine kinase, has multifactorial roles for controlling cell proliferation, differentiation, motility, inflammation, and apoptosis (Jope and Johnson, 2004). Overexpression of GSK-3 or blockage of phosphatidylinositol 3-kinase/Akt, the negative regulator of GSK-3 , causes apoptosis (Pap and Cooper, 1998). GSK-3 is also involved in ER stress-induced apoptosis (Chen et al., 2004; Brewster et al., 2006); however, the exact downstream targets of GSK-3 remain inconclusive. GSK-3 can phosphorylate Bax and cause its conformational change (Linseman et al., 2004). In growth factor withdrawalinduced apoptosis, GSK-3 causes Mcl-1 phosphorylation followed by degradation via an ubiquitin-proteasome system (Maurer et al., 2006). Current studies show that caspase-2 mediates proapoptotic Bax and Bid activation during genotoxic stimulation and ER stress, respectively (Cao et al., 2008; Upton et al., 2008). We previously showed that GSK-3 is essential for ceramideand etoposide-induced caspase-2 activation before mitochondrial apoptosis (Lin et al., 2007). In the present study, we found an essential role of GSK-3 in ER stress-induced lysosomal apoptosis. GSK-3 regulated initial caspase-2 activation upstream of lysosomal destabilization but was mitochondria independent. Furthermore, lysosomal cathepsin B-mediated caspase-8 and -3 activation was demonstrated. Materials and Methods Cell Culture and Reagents. Human monocytic leukemia/lymphoma U937, acute myeloid leukemia HL-60, and chronic myeloid leukemia K562 cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum and maintained at 37°C in 5% CO2. Ceramide analog C2-ceramide (BIOMOL Research Laboratories, Plymouth Meeting, PA), tunicamycin, Z-FA-fmk, Isovalery-Val-Val-Sta-Ala-Sta (pepstatin A), SB415286, and LY294002 (Sigma-Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide (DMSO). 4,6-Diamidino-2-phenylindole (DAPI), propidium iodide (PI), and acridine orange (AO) were purchased from Sigma-Aldrich. Caspase-2 inhibitor Z-VDVADfmk, caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, caspase-4 inhibitor Z-Leu-Glu(Ome)-ValAsp(Ome)-fluoromethyl ketone, caspase-8 inhibitor Z-IETD-fmk, caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone, and broad-spectrum caspase inhibitor Z-VAD-fmk were dissolved in DMSO and purchased from Sigma-Aldrich. Western Blotting. To detect indicated proteins, total cell lysates were extracted using a Triton X-100-based lysis buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.5, 5 mM EDTA, 5 mM NaN3, 10 mM NaF, and 10 mM sodium pyrophosphate with a protease inhibitor mix and a phosphatase inhibitor cocktail I (Sigma-Aldrich)]. Proteins were separated using SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). After blocking, blots were developed with a series of antibodies as indicated. Antibodies specific for caspase-2, -8, and -9 and GRP78, GSK-3 , and phosphoGSK-3 (Ser9) were purchased from Cell Signaling Technology Inc. (Danvers, MA); antibodies specific for caspase-3, CHOP, and phospho-GSK-3 (Tyr216) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and antibodies against caspase-4 and -actin were from Sigma-Aldrich. Finally, blots were hybridized with horseradish peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology Inc.) and developed using an ECL Western blot detection kit (Millipore Corporation) according to the manufacturer’s instructions. Analysis of Apoptosis. For apoptosis detection, cells were fixed with 70% ethanol in phosphate-buffered saline (PBS) for PI staining and then were analyzed using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA). Apoptotic cell membrane disruption characterized by the presence of phosphatidylserine was performed using an annexin V-fluorescein isothiocyanate detection kit (BioVision, Mountain View, CA), followed by flow cytometric analysis and fluorescence microscopic observation (BX51; Olympus, Tokyo, Japan). DAPI and Hoechst 33342 dye were used for nuclear staining. Analysis of Caspase Activity. For the detection of caspase-2 activation, a caspase-2 colorimetric assay kit (Millipore Bioscience Research, Temecula, CA) was used according to the manufacturer’s instructions. Optical density measurements were performed using a Fluorskan Ascent fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA). Short Interfering RNA and Plasmid Transfection. The short interfering RNAs (siRNAs) specific for human caspase-2 and GSK-3 were purchased from Santa Cruz Biotechnology, Inc. The pcDNA 3.1 constructs expressing a constitutively active form of GSK-3 (S9A) was a gift from Dr. P. J. Lu (Institute of Clinical Medicine, National Cheng Kung University Medical College, Taiwan). Transient transfection was performed using an MP-100 Microporator (Digital Biotechnology, Seoul, Korea) according to the manufacturer’s instructions for optimization and usage. After transfection, the cells were cultured for 24 h before the experiments. Assay of LMP. Cell cultures were vitally stained with AO at a concentration of 5 g/ml for 15 min and then washed with PBS. AO is a lysosomotropic weak base and a concentration-dependent metachromatic fluorophore. AO relocation from lysosomes to cytosol, and the decrease of granular (lysosomal) red fluorescence (dimer form) in ER Stress Induces Lysosomal Apoptosis through GSK-3 525 at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from combination with the increased diffuse (cytosolic and nuclei) green fluorescence (monomer form) may imply the deterioration of lysosomal membrane stability with a decreased proton gradient, which permits the leakage of the lysosomal contents to cytosol. The levels of LMP were analyzed using flow cytometry (FACSCalibur) with excitation set at 488 nm (FL-3). In addition, the relative intensities of red and green fluorescence were simultaneously examined using fluorescence microscopy (BX51). Immunostaining. For intracellular immunostaining, cells were fixed and permeabilized with 4% formaldehyde in PBS for 10 min at room temperature. After the cells were washed, anti-cathepsin B antibody (Calbiochem, San Diego, CA) was used followed by FITCconjugated secondary antibody (BioVision) staining. DAPI was used for nuclear staining. Statistical Analysis. Values are as means S.D. Groups were compared using Student’s two-tailed unpaired t test or one-way analysis of variance analysis. Statistical significance was set at p 0.05.