In-Ki Kim

Chemical Biology Investigation of Cell Death Pathways Activated by Endoplasmic Reticulum Stress Reveals Cytoprotective Modulators of ASK1

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.

Pro-apoptotic function of calsenilin/DREAM/KChIP3

Apoptotic cell death and increased production of amyloid β peptide (Aβ) are pathological features of Alzheimer’s disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro‐apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide‐induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+‐ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3‐induced apoptosis was suppressed by caspase inhibitor z‐VAD and by Bcl‐xL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPsw) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C‐terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Aβ42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis‐associated Aβ42 production of calsenilin/DREAM/KChIP3.

The involvement of oxidative stress in tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HeLa cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal triggering apoptosis in tumor cells. However, the molecular mechanisms leading to the apoptosis are largely unknown. To characterize the molecular events involved in TRAIL-induced apoptosis, we examined the association of reactive oxygen species (ROS) in human adenocarcinoma HeLa cells. In this study, we show strong ROS accumulation upon TRAIL induction, with activation of caspases, followed by apoptosis. The pre-treatment with gamma-glutamylcysteinylglycine or estrogen, both effective antioxidants, significantly attenuated TRAIL-induced apoptosis through the reduction of ROS accumulation and diminished caspases activity. Furthermore, zVAD-fmk, an inhibitor of pan-caspase, effectively inhibited the activation of caspases and prevented apoptosis by TRAIL, although TRAIL-induced ROS generation was not attenuated. These data indicate that ROS may play a role as an upstream mediator of caspases. Taken together, our results suggest that oxidative stress mediates TRAIL-induced apoptosis in HeLa cells.

Functional screening of genes suppressing TRAIL-induced apoptosis: distinct inhibitory activities of Bcl-XL and Bcl-2

TNF-related apoptosis-inducing ligand (TRAIL) is known to selectively induce apoptosis in various tumour cells. However, downstream-signalling of TRAIL-receptor is not well defined. A functional genetic screening was performed to isolate genes interfering with TRAIL-induced apoptosis using cDNA retroviral library. Bcl-XL and FLIP were identified after DNA sequencing analysis of cDNA rescued from TRAIL-resistant clones. We found that increased expression of Bcl-XL, but not Bcl-2, suppressed TRAIL-induced apoptosis in tumour cells. Western blot and immunohistochemical analyses showed that expression of Bcl-XL, but not Bcl-2, was highly increased in human breast cancer tissues. Exposure of MDA-MB-231 breast tumour cells to TRAIL induced apoptosis accompanied by dissipation of mitochondrial membrane potential and enzymatic activation of caspase-3, -8, and -9. However, SK-BR-3 breast tumour cells exhibiting increased expression level of Bcl-XL were resistant to TRAIL, though upon exposure to TRAIL, caspase-8 and Bid were activated. Forced expression of Bcl-XL, but not Bcl-2, desensitised TRAIL-sensitive MDA-MB-231 cells to TRAIL. Similar inhibitory effects were also observed in other tumour cells such as HeLa and Jurkat cells stably expressing Bcl-XL, but not Bcl-2. These results are indicative of the crucial and distinct function of Bcl-XL and Bcl-2 in the modulation of TRAIL-induced apoptosis.

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.

The roles of ER stress and P450 2E1 in CCl4-induced steatosis

The role of ER stress on hepatic steatosis was investigated in a rat model. We injected CCl(4) into rats and found that CCl(4) could induce hepatic lipid accumulation, confirmed by Oil Red O staining and by measurement of triglyceride and cholesterol. The expression of ApoB, an apolipoprotein, was decreased in plasma and increased in the liver of CCl(4)-treated animals. The ER stress response was also significantly increased by CCl(4). P450 2E1 expression and activity were increased through interactions of P450 2E1 with NADPH-dependent P450 reductase (NPR) under CCl(4)-treated conditions. In HepG2 cells, intracellular lipid accumulation and its signaling were comparable to in vivo results. In order to elucidate the effect of the ER stress response itself, tunicamycin, an N-acetyl-glycosylation inhibitor, was injected into rats, followed by Oil Red O staining, lipid/triglyceride/cholesterol accumulation analysis, and examination of ApoB expression. Additionally, the ER stress response and upregulation of P450 2E1 were also confirmed in the tunicamycin-treated rats. All of the responses were similar to those seen with CCl(4). The P450 2E1 inhibitor diallyl sulphide (DAS), N-acetylcysteine (NAC), and reduced glutathione (GSH) antioxidants also regulated processes, including ApoB expression and lipid accumulation in CCl(4)-treated animals. In the presence of tunicamycin, DAS or NAC/GSH regulated all of the pathological phenomena with the exception of the ER stress response. In summary, CCl(4) induces liver steatosis, a process involving ER stress-induced P450 2E1 activation and ROS production.

Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53

Osteopontin (OPN) is highly expressed in cancer patients and plays important roles in many stages of tumor progression, such as anti-apoptosis, proliferation, and metastasis. From functional screening of human cDNA library, we isolated OPN as a caspase-8 substrate that regulates cell death during hypoxia/reoxygenation (Hyp/RO). In vitro cleavage assays demonstrate that OPN is cleaved at Asp-135 and Asp-157 by caspase-8. Cellular cleavage of OPN is observed in apoptotic cells exposed to Hyp/RO among various apoptotic stimuli and its cleavage is blocked by zVAD or IETD caspase inhibitor. Further, over-expression of OPN, the form with secretion signal, inhibits Hyp/RO-induced cell death. Caspase cleavage-defective OPN mutant (OPN D135A/D157A) is more efficient to suppress Hyp/RO-induced cell death than wild-type OPN. OPN D135A/D157A sustains AKT activity to increase cell viability through inhibition of caspase-9 during Hyp/RO. In addition, OPN is highly induced in some tumor cells during Hyp/RO, such as HeLa and Huh-7 cells, which is associated with their resistance to Hyp/RO by sustaining AKT activity. Notably, OPN C-terminal cleavage fragment produced by caspase-8 is detected in the nucleus. Plasmid-encoded expression of OPN C-terminal cleavage fragment increases p53 protein level and induces apoptosis of wild-type mouse embryonic fibroblast cells, but not p53−/− mouse embryonic fibroblast cells. These observations suggest that the protective function of OPN during Hyp/RO is inactivated via the proteolytic cleavage by caspase-8 and its cleavage product subsequently induces cell death via p53, postulating caspase-8 as a negative regulator of tumorigenic activity of OPN.

Caspase cleavage product lacking amino-terminus of IκBα sensitizes resistant cells to TNF-α and TRAIL-induced apoptosis

In response to a diverse array of signals, IκBα is targeted for phosphorylation‐dependent degradation by the proteasome, thereby activating NF‐κB. Here we demonstrate a role of the cleavage product of IκBα in various death signals. During apoptosis of NIH3T3, Jurkat, Rat‐1, and L929 cells exposed to tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL), Fas, serum deprivation, or TNF‐α, respectively, IκBα was cleaved in a caspase‐dependent manner. In vitro and in vivo cleavage assays and site‐directed mutagenesis showed that caspase‐3 cleaved IκBα between Asp31 and Ser32. Expression of the cleavage product lacking amino‐terminus (1–31), ΔIκBα, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF‐α or TRAIL, and HeLa tumor cells to TNF‐α. ΔIκBα was more pro‐apoptotic compared to wild type or cleavage‐resistant (D31E)IκBα mutant and the sensitization elicited by ΔIκBα was as effective as that by the dominant negative mutant, (S32,36A)IκBα, in NIH3T3 cells. ΔIκBα suppressed the transactivation of NF‐κB induced by TNF‐α or TRAIL, as reflected by luciferase‐reporter activity. Conversely, expression of the p65 subunit of NF‐κB suppressed TNF‐α‐, TRAIL‐, and serum deprivation‐induced cell death. On the contrary, ΔIκBα was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that ΔIκBα generated by various death signals sensitizes cells to apoptosis by suppressing NF‐κB activity. J. Cell. Biochem. 85: 334–345, 2002.

Inactivation of farnesyltransferase and geranylgeranyltransferase I by caspase-3: Cleavage of the common α subunit during apoptosis

Caspase plays an important role in apoptosis. We report here that farnesyltransferase/geranylgeranyltransferase (FTase/GGTase)-α, a common subunit of FTase (α/βFTase) and GGTase I (α/βGGTase), was cleaved by caspase-3 during apoptosis. FTase/GGTase-α (49 kDa) was cleaved to 35 kDa (p35) in the Rat-2/H-ras, W4 and Rat-1 cells treated with FTase inhibitor (LB42708), anti-Fas antibody and etoposide, respectively. This cleavage was inhibited by caspase-inhibitors (YVAD-cmk, DEVD-cho). Serial N-terminal deletions and site-directed mutagenesis showed that Asp59 of FTase/GGTase-α was cleaved by caspase-3. The common FTase/GGTase-α subunit, but not the β subunits, of the FTase or GGTase I protein complexes purified from baculovirus-infected SF-9 cells was cleaved to be inactivated by purified caspase-3. In contrast, FTase mutant protein complex [(D59A)α/βFTase] was resistant to caspase-3. Expression of either the cleavage product (60-379) or anti-sense of FTase/GGTase-α induced cell death in Rat-2/H-ras cells. Furthermore, expression of (D59A)FTase/GGTase-α mutant significantly desensitized cells to etoposide-induced death. Taken together, we suggest that cleavage of prenyltransferase by caspase contributes to the progression of apoptosis.

Enhanced Lysosomal Activity Is Involved in Bax Inhibitor-1-induced Regulation of the Endoplasmic Reticulum (ER) Stress Response and Cell Death against ER Stress INVOLVEMENT OF VACUOLAR H+-ATPASE (V-ATPASE)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H+-ATPase(V-ATPase), a proton pump, was activated, suggesting high H+ uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.