Wei-Ching Huang

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

The signaling of glycogen synthase kinase-3β (GSK-3β) has been implicated in stress-induced apoptosis. However, the pro-apoptotic role of GSK-3β is still unclear. Here, we show the involvement of GSK-3β in ceramide-induced mitochondrial apoptosis. Ceramide induced GSK-3β activation via protein dephosphorylation at serine 9. We previously reported that ceramide induced caspase-2 and caspase-8 activation, Bid cleavage, mitochondrial damage, and apoptosis. In this study, we found that caspase-2 activation and the subsequent apoptotic events were abolished by the GSK-3β inhibitors lithium chloride and SB216763, and by GSK-3β knockdown using short interfering RNA. We also found that ceramide-activated protein phosphatase 2A (PP2A) indirectly caused GSK-3β activation, and that the PP2A-regulated PI 3-kinase-Akt pathway was involved in GSK-3β activation. These results indicate a role for GSK-3β in ceramide-induced apoptosis, in which GSK-3β acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 and caspase-8.

Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells.

The inflammatory effects of glycogen synthase kinase‐3 (GSK‐3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK‐3‐mediated interleukin‐10 (IL‐10) inhibition on lipopolysaccharide (LPS)‐induced inflammation. Treatment with GSK‐3 inhibitor significantly blocked LPS‐induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia‐enriched cultures. Using an antibody array and enzyme‐linked immunosorbent assay, we found that GSK‐3‐inhibitor treatment blocked LPS‐induced upregulation of regulated on activation normal T‐cell expressed and secreted (RANTES) and increased IL‐10 expression. The time kinetics and dose–response relations were confirmed. Reverse transcription–polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK‐3 using short‐interference RNA, and transfecting cells with dominant‐negative GSK‐3β, blocked LPS‐elicited NO and RANTES expression but increased IL‐10 expression. In contrast, GSK‐3β overexpression upregulated NO and RANTES but downregulated IL‐10 in LPS‐stimulated cells. Treating cells with anti‐IL‐10 neutralizing antibodies to prevent GSK‐3 from downregulating NO and RANTES showed that the anti‐inflammatory effects are, at least in part, IL‐10‐dependent. The involvement of Akt, extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase and nuclear factor‐κB that positively regulated IL‐10 was demonstrated. Furthermore, inhibiting GSK‐3 increased the nuclear translocation of transcription factors, that all important for IL‐10 expression, including CCAAT/enhancer‐binding protein beat (C/EBPβ), C/EBPδ, cAMP response binding element protein and NF‐κB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK‐3‐mediated IL‐10 downregulation.

Vinca alkaloids cause aberrant ROS-mediated JNK activation, Mcl-1 downregulation, DNA damage, mitochondrial dysfunction, and apoptosis in lung adenocarcinoma cells

Vinca alkaloids are clinically used to inhibit the growth of malignancy by interfering with microtubule polymerization. The purpose of this study was to identify the molecular mechanisms underlying growth inhibition as well as apoptosis in vinca alkaloid-treated lung adenocarcinoma cells. Consistent with nocodazole, treatment with vinorelbine (VNR) caused mitotic prometaphase arrest in a time-dependent manner, accompanied by cell apoptosis, dependent on both dose and time. VNR sequentially induced mitochondrial transmembrane potential (MTP) loss and caspase-dependent apoptosis following myeloid cell leukemia (Mcl) 1 downregulation. Prolonged activation of c-Jun N-terminal kinase (JNK) was required for vinca alkaloid- and nocodazole-induced apoptosis but not cell cycle arrest. Vinca alkaloids and nocodazole caused glutathione/reactive oxygen species (ROS) imbalance, and inhibiting ROS prevented prolonged JNK activation, decreased Mcl-1 levels, MTP loss, and apoptosis. Notably, cell size and granularity were enlarged in stimulated cells; unexpectedly, many ROS-producing mitochondria were accumulated followed by aberrant JNK-mediated mitochondrial dysfunction. Unlike cisplatin, which causes DNA damage in each phase of the cell cycle, VNR and nocodazole induced aberrant JNK-regulated DNA damage in prometaphase; however, inhibiting ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3-related) did not reverse mitotic arrest or apoptosis. These results demonstrate an essential role of ROS in vinca alkaloid-induced aberrant JNK-mediated Mcl-1 downregulation and DNA damage followed by mitochondrial dysfunction-related apoptosis but not mitotic arrest.

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

Glycogen synthase kinase (GSK)-3β 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-3β 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-3β and caspase-2 in response to tunicamycin. Activating GSK-3β caused K562 cells to be susceptible to tunicamycin-induced apoptosis. Taken together, we show that GSK-3β 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.

Inhibiting glycogen synthase kinase-3 reduces endotoxaemic acute renal failure by down-regulating inflammation and renal cell apoptosis

Background and purpose:u2002 Excessive inflammation and apoptosis are pathological features of endotoxaemic acute renal failure. Activation of glycogen synthase kinase‐3 (GSK‐3) is involved in inflammation and apoptosis. We investigated the effects of inhibiting GSK‐3 on lipopolysaccharide (LPS)‐induced acute renal failure, nuclear factor‐κB (NF‐κB), inflammation and apoptosis.

Staphylococcus aureus induces microglial inflammation via a glycogen synthase kinase 3β-regulated pathway

ABSTRACT A proinflammatory role for glycogen synthase kinase 3β (GSK-3β) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-α largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3β, and inhibiting GSK-3β reduced TNF-α production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-κB was essential for heat-inactivated S. aureus-induced TNF-α and NO, inhibiting GSK-3β blocked heat-inactivated S. aureus-induced NF-κB p65 nuclear translocation. Additionally, inhibiting GSK-3β enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-α production). Neutralization of IL-10 reduced TNF-α downregulation caused by GSK-3β inhibition. These results suggest that GSK-3β regulates heat-inactivated S. aureus-induced TNF-α and NO production in microglia mainly by activating NF-κB and probably by inhibiting IL-10.

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer.

Oncogenic activation accompanied by escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.

Glycogen synthase kinase-3β indirectly facilitates interferon-γ-induced nuclear factor-κB activation and nitric oxide biosynthesis.

Either glycogen synthase kinase (GSK)‐3β or nuclear factor (NF)‐κB regulates interferon (IFN)‐γ‐induced nitric oxide (NO) biosynthesis; however, the inter‐regulation between GSK‐3β and NF‐κB is unknown. We have previously shown that IFN‐γ‐activated GSK‐3β negatively regulates Src homology‐2 domain‐containing phosphatase (SHP) 2 to facilitate Janus kinase (Jak) 2‐signal transducer and activator of transcription (STAT) 1 activation. Because Jaks‐IFN‐inducible dsRNA‐activated serine–threonine protein kinase (PKR) axis signaling is essential for IFN‐γ‐activation of NF‐κB, in this study we investigate the potential mechanism for GSK‐3β‐facilitated NF‐κB activation in IFN‐γ‐stimulated RAW264.7 murine macrophages. Pharmacological inhibitors of GSK‐3β or NF‐κB signaling, such as the inhibitor of κB (IκB) kinase β (IKKβ) and IκBα, inhibited IFN‐γ‐induced inducible NO synthase (iNOS) and thus NO biosynthesis. Inhibiting GSK‐3β decreased IFN‐γ‐induced NF‐κB phosphorylation (Ser536) and activation. The upstream regulators for GSK‐3β activation, including okadaic acid‐sensitive protein phosphatase and proline‐rich tyrosine kinase 2, were also important for IFN‐γ‐induced IκBα phosphorylation (Ser32) and degradation. Under IFN‐γ stimulation, Jak2–PKR axis signaling induced IκBα inactivation as well as iNOS/NO biosynthesis. It is notable that inhibiting GSK‐3β caused SHP2‐mediated dephosphorylation of PKR (Thr446), IKKβ (Ser180), and NF‐κB (Ser536). Taken together, we provide the first evidence to demonstrate that GSK‐3β indirectly facilitates IFN‐γ‐induced NF‐κB activation by inhibiting SHP2, in turn activating the PKR–IKKβ–IκBα axis signaling pathway. J. Cell. Biochem. 111: 1522–1530, 2010.

Different Types of Cell Death Induced by Enterotoxins

The infection of bacterial organisms generally causes cell death to facilitate microbial invasion and immune escape, both of which are involved in the pathogenesis of infectious diseases. In addition to the intercellular infectious processes, pathogen-produced/secreted enterotoxins (mostly exotoxins) are the major weapons that kill host cells and cause diseases by inducing different types of cell death, particularly apoptosis and necrosis. Blocking these enterotoxins with synthetic drugs and vaccines is important for treating patients with infectious diseases. Studies of enterotoxin-induced apoptotic and necrotic mechanisms have helped us to create efficient strategies to use against these well-characterized cytopathic toxins. In this article, we review the induction of the different types of cell death from various bacterial enterotoxins, such as staphylococcal enterotoxin B, staphylococcal alpha-toxin, Panton-Valentine leukocidin, alpha-hemolysin of Escherichia coli, Shiga toxins, cytotoxic necrotizing factor 1, heat-labile enterotoxins, and the cholera toxin, Vibrio cholerae. In addition, necrosis caused by pore-forming toxins, apoptotic signaling through cross-talk pathways involving mitochondrial damage, endoplasmic reticulum stress, and lysosomal injury is discussed.