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1.  Interaction between Neuronal Depolarization and MK-801 in SH-SY5Y Cells and the Rat Cortex 
Psychiatry Investigation  2008;5(2):94-101.
The interaction between MK-801, a model of psychosis and KCl-induced depolarization or electroconvulsive shock (ECS), a therapeutic model of electroconvulsive therapy (ECT), was investigated in SH-SY5Y cells and the rat frontal cortex.
SH-SY5Y cells were pretreated with 1 µM MK-801 for 15 min, followed by cotreatment with 100 mM KCl for 5 min. MK-801 was reintroduced after the KCl was washed out, and the samples were incubated before harvesting. For the experiments in rats, male Sprague-Dawley rats were treated with MK-801 followed by ECS. Immunoblot analyses of glycogen synthase kinase 3β (GSK3β) (Ser9), AKT (Ser473) and extracellular legulated kinase (ERK)1/2 in SH-SY5Y cells and the rat frontal cortex were performed.
KCl-induced neuronal depolarization resulted in the transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), followed by increased phosphorylation of the enzymes in SH-SY5Y cells. Cotreatment with MK-801 and KCl inhibited the initial dephosphorylation of AKT and GSK3β produced by KCl-induced neuronal depolarization. Similarly, ECS resulted in the transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), whereas cotreatment with MK-801 inhibited the initial dephosphorylation of AKT (Ser473) and GSK3β (Ser9) produced by ECS in the rat frontal cortex. No significant interaction was observed between MK-801 and KCl in the dephosphorylation of ERK1/2.
These results suggest that an antagonistic interplay between MK-801 and neuronal depolarization by KCl or ECS is involved the regulation of AKT (Ser473) and GSK3β (Ser9) phosphorylation.
PMCID: PMC2796014  PMID: 20046351
AKT; Glycogen synthase kinase 3β; Extracellular legulated kinase 1/2; KCl; Electro convulsive shock; MK-801
2.  Cadmium induces neuronal cell death through reactive oxygen species activated by GADD153 
BMC Cell Biology  2013;14:4.
Cadmium(Cd), a heavy metal, which has a potent harmful effects, is a highly stress-inducible material that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). The mechanism Cd induced cell death of neuroblastoma cells complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). Several studies have correlated GADD153 expression with cell death, but a mechanistic link between GADD153 and apoptosis has never been demonstrated.
SH-SY5Y cells were treated Cd led to increase in intracellular ROS levels. ROS generation is not consistent with intracellular [Ca2+]. The exposure of neuroblastoma cells to Cd led to increase in intracellular GADD153 and Bak levels in a doses and time dependent manner. The induction of these genes by Cd was attenuated by NAC. Cd-induced apoptosis is decreased in GADD153 knockdown cells compared with normal cells. The effect of GADD153 on the binding of C/EBP to the Bak promoters were analyzed ChIP assay. Basal constitutive GADD153 recruitment to the –3,398/–3,380 region of the Bak promoter is observed in SH-SY5Y cells.
The exposure of SH-SY5Y cells to Cd led to increase in intracellular ROS levels in a doses and time dependent manner. The generation of ROS result in the induction of GADD153 is causative of cadmium-induced apoptosis. GADD153 regulates Bak expression by its binding to promoter region (between −3,398 and −3,380). Therefore, we conclude that GADD153 sensitizes cells to ROS through mechanisms that involve up-regulation of BAK and enhanced oxidant injury.
PMCID: PMC3563515  PMID: 23339468
Cadmium; ROS; ER; Bak
3.  Potential role and mechanism of IFN-gamma inducible protein-10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in rheumatoid arthritis 
Arthritis Research & Therapy  2011;13(3):R104.
IFN-gamma inducible protein-10 (CXCL10), a member of the CXC chemokine family, and its receptor CXCR3 contribute to the recruitment of T cells from the blood stream into the inflamed joints and have a crucial role in perpetuating inflammation in rheumatoid arthritis (RA) synovial joints. Recently we showed the role of CXCL10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in an animal model of RA and suggested the contribution to osteoclastogenesis. We tested the effects of CXCL10 on the expression of RANKL in RA synoviocytes and T cells, and we investigated which subunit of CXCR3 contributes to RANKL expression by CXCL10.
Synoviocytes derived from RA patients were kept in culture for 24 hours in the presence or absence of TNF-α. CXCL10 expression was measured by reverse transcriptase polymerase chain reaction (RT-PCR) of cultured synoviocytes. Expression of RANKL was measured by RT-PCR and western blot in cultured synoviocytes with or without CXCL10 and also measured in Jurkat/Hut 78 T cells and CD4+ T cells in the presence of CXCL10 or dexamethasone. CXCL10 induced RANKL expression in Jurkat T cells was tested upon the pertussis toxin (PTX), an inhibitor of Gi subunit of G protein coupled receptor (GPCR). The synthetic siRNA for Gαi2 was used to knock down gene expression of respective proteins.
CXCL10 expression in RA synoviocytes was increased by TNF-α. CXCL10 slightly increased RANKL expression in RA synoviocytes, but markedly increased RANKL expression in Jurkat/Hut 78 T cell or CD4+ T cell. CXCL10 augmented the expression of RANKL by 62.6%, and PTX inhibited both basal level of RANKL (from 37.4 ± 16.0 to 18.9 ± 13.0%) and CXCL10-induced RANKL expression in Jurkat T cells (from 100% to 48.6 ± 27.3%). Knock down of Gαi2 by siRNA transfection, which suppressed the basal level of RANKL (from 61.8 ± 17.9% to 31.1 ± 15.9%) and CXCL10-induced RANKL expression (from 100% to 53.1 ± 27.1%) in Jurkat T cells, is consistent with PTX, which inhibited RANKL expression.
CXCL10 increased RANKL expression in CD4+ T cells and it was mediated by Gαi subunits of CXCR3. These results indicate that CXCL10 may have a potential role in osteoclastogenesis of RA synovial tissue and subsequent joint erosion.
PMCID: PMC3218919  PMID: 21708014
4.  Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells 
Experimental & Molecular Medicine  2009;41(8):592-600.
Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the α subunit of Gs (GαsQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GαsQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GαsQL-stimulated Bak reporter luciferase activity. Expression of GαsQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Gαs activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Gαs augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.
PMCID: PMC2739899  PMID: 19381065
apoptosis; bcl-2 homologous antagonist-killer protein; cyclic AMP; gamma rays; heterotrimeric GTP-binding protein; receptors, G-protein-coupled
5.  STAT3 inhibits the degradation of HIF-1α by pVHL-mediated ubiquitination 
Experimental & Molecular Medicine  2008;40(5):479-485.
Hypoxia-inducible factor 1α (HIF-1α) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1α is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1α protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1α stability. We found that STAT3 interacts with C-terminal domain of HIF-1α and stabilizes HIF-1α by inhibition of pVHL binding to HIF-1α. The binding between HIF-1α and pVHL, negative regulator of HIF-1α stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1α protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1α protein via inhibition of interaction between pVHL and HIF-1α. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1α through competition with pVHL for binding to HIF-1α, and then stabilizes HIF-1α protein levels.
PMCID: PMC2679355  PMID: 18985005
anoxia; hypoxia-inducible factor1, α subunit; neoplasms; STAT3 transcription factor; ubiquitination; von Hippel-Lindau tumor suppressor protein
6.  Repeated electroconvulsive seizure induces c-Myc down-regulation and Bad inactivation in the rat frontal cortex 
Experimental & Molecular Medicine  2008;40(4):435-444.
Repeated electroconvulsive seizure (ECS), a model for electroconvulsive therapy (ECT), exerts neuroprotective and proliferative effects in the brain. This trophic action of ECS requires inhibition of apoptotic activity, in addition to activation of survival signals. c-Myc plays an important role in apoptosis of neurons, in cooperation with the Bcl-2 family proteins, and its activity and stability are regulated by phosphorylation and ubiquitination. We examined c-Myc and related proteins responsible for apoptosis after repeated ECS. In the rat frontal cortex, repeated ECS for 10 days reduced the total amount of c-Myc, while increasing phosphorylation of c-Myc at Thr58, which reportedly induces degradation of c-Myc. As expected, ubiquitination of both phosphorylated and total c-Myc increased after 10 days ECS, suggesting that ECS may reduce c-Myc protein level via ubiquitination-proteasomal degradation. Bcl-2 family proteins, caspase, and poly(ADP-ribose) polymerase (PARP) were investigated to determine the consequence of down-regulating c-Myc. Protein levels of Bcl-2, Bcl-XL, Bax, and Bad showed no change, and cleavage of caspase-3 and PARP were not induced. However, phosphorylation of Bad at Ser-155 and binding of Bad to 14-3-3 increased without binding to Bcl-XL after repeated ECS, implying that repeated ECS sequesters apoptotic Bad and frees pro-survival Bcl-XL. Taken together, c-Myc down-regulation via ubiquitination-proteasomal degradation and Bad inactivation by binding to 14-3-3 may be anti-apoptotic mechanisms elicited by repeated ECS in the rat frontal cortex. This finding further supports the trophic effect of ECS blocking apoptosis as a possible therapeutic effect of ECT.
PMCID: PMC2679266  PMID: 18779656
apoptosis; bcl-2-associated X protein; electroconvulsive therapy; nerve growth factors; proto-oncogene proteins c-bcl-2; proto-oncogene proteins c-myc; ubiquitination

Results 1-6 (6)