Anti-apoptotic action of (2S,3S,4R)-N″-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine (KR-31378) by suppression of the phosphatase and tensin homolog deleted from chromosome 10 phosphorylation and increased phosphorylation of casein kinase2/Akt/ cyclic AMP response element binding protein via maxi-K channel opening in neuronal cells

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Abstract

This study shows the signaling pathway by which (2S,3S,4R)-N″-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine (KR-31378) prevents tumor necrosis factor (TNF)-α-induced neuronal cell death. KR-31378 restored TNF-α-induced decreased cell viability of SK–N–SH. U87-MG cells (PTEN-null glioblastoma cell line) transfected with expression vectors for sense PTEN (phosphatase and tensin homolog deleted from chromosome 10) showed significantly decreased cell viability, which was restored by KR-31378. TNF-α-induced increased PTEN phosphorylation and decreased phosphorylation of Akt/cyclic AMP response element-binding protein (CREB) in SK–N–SH cells were concentration-dependently reversed by KR-31378, those of which were antagonized by iberiotoxin, a maxi-K channel blocker. TNF-α and apigenin, a casein kinase2 (CK2) inhibitor, showed decreased CK2 phosphorylation and increased PTEN phosphorylation, which were reversed by KR-31378. KR-31378 increased K+ currents by activating the maxi-K channels in SK–N–SH cells, with suppression of TNF-α-induced increase in cytosolic Ca2+ and elevation of suppressed mitochondrial membrane potential, all of which were antagonized by iberiotoxin. It is suggested that increase in cell viability by KR-31378 is ascribed to the maxi-K channel opening-coupled upregulation of CK2/Akt/CREB phosphorylation and downregulation of PTEN phosphorylation in association with increased Bcl-2 and decreased Bax levels.

Introduction

Recently, it has been demonstrated that (2S,3S,4R)-N″-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine (KR-31378) potently suppressed lipopolysaccharide-induced cell death and DNA fragmentation in human umbilical vein endothelial cells in association with significant reduction in the intracellular reactive oxygen species and tumor necrosis factor (TNF)-α. Lipopolysaccharide-induced decrease in Bcl-2 and increase in Bax protein and cytochrome c release were totally reversed by KR-31378 (Kim et al., 2002). Hong et al. (2002) have shown the in vivo results, in that the infarct area of rats subjected to 2 h occlusion of the left middle cerebral artery followed by 24 h reperfusion was significantly reduced after treatment with KR-31378, after the completion of 2 h ischemia. Treatment with KR-31378 significantly reduced the TUNEL-positive cells and suppressed the DNA fragmentation in the cortical tissue associated with increased Bcl-2 protein and decreased Bax protein and cytochrome c release.

PTEN (the phosphatase and tensin homolog deleted from chromosome 10) that has both protein phosphatase (phospho-serine/threonine and phospho-tyrosine) and phosphoinositide 3-phosphatase activities (Myers et al., 1997, Maehama and Dixon, 1998) negatively regulates the phosphatidylinositol 3-kinase by catalyzing degradation of the phosphatidylinositol(3,4,5)-triphosphate (PI(3,4,5)P3) to phosphatidylinositol(3,4)-diphosphate (PI(3,4)P2) (Stambolic et al., 1998). Evidence has accumulated that a signaling cascade mediated by activated Akt/cyclic AMP response element-binding protein (CREB) regulates Bcl-2 expression and promotes cell survival by growth factors against apoptotic stimuli (Dudek et al., 1997, Kulik et al., 1997). Overexpression of phosphatidylinositol 3-kinase and its downstream effector Akt (serine/threonine kinase) mediate growth factor-induced neuronal survival, and they in turn up-regulate Bcl-2 promoter activity, in association with Bcl-2 protein expression through enhanced CREB activation (Crowder and Freeman, 1998, Walton et al., 1999, Pugazhenthi et al., 2000). Protein kinase CK2 (CK2, formerly known as casein kinase 2) has a role in the regulation of cell growth and proliferation, catalysis of the phosphorylation of a number of proteins and in the modulation of the activities of proteins (Pinna, 1990, Allende and Allende, 1995).

On the other hand, maxi-K channels, a large conductance Ca2+-activated K+ channels, are activated by depolarization and increase in intracellular Ca2+ (Latorre et al., 1989). Gribkoff et al. (2001) introduced the usefulness of the maxi-K channel opener, (3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one (BMS 204352), for neuroprotection against acute ischemic stroke by restricting Ca2+ entry in neurons at risk. Robitaille and Charlton (1992) early demonstrated the limited accumulation of pathological levels of Ca2+ by K+ channel opening during brain ischemia, thereby reducing the neurotransmitter release and attenuating the ischemic injury. Most recently, Rundén-Pran et al. (2002) showed a protective role of maxi-K channels, in that treatment with maxi-K channel blocker, paxilline (a mycotoxin naturally produced by the fungus Penicillium) and iberiotoxin (a peptidyl scorpion toxin) enhanced cell death of the hippocampus during and after oxygen–glucose deprivation. However, little information is known regarding the relationships between activation of maxi-K channels and regulation of PTEN phosphorylation (P-PTEN) and/or CK2/Akt/CREB phosphorylation (P-CK2, P-Akt, P-CREB) in the cell viability.

In the present study, to elucidate the signaling pathway by which KR-31378 increases the cell viability, we examined the protective effect of KR-31378 against TNF-α-induced reduction in viability in the SK–N–SH cells (neuroblastoma cells). To verify implication of PTEN, we employed U87-MG cells (PTEN-null glioblastoma cell line) and U87-MG cells transfected with expression vectors for sense PTEN (sPTEN). To further identify the mechanism by which KR-31378 ameliorates cell injury, we determined the changes in P-CK2, P-PTEN, P-Akt, P-CREB, Bcl-2 and Bax protein levels by Western blot analysis under treatment with KR-31378, in the absence and presence of iberiotoxin, a maxi-K channel blocker. Finally, we electrophysiologically confirmed the effect of KR-31378 on the K+ current in SK–N–SH cells.

Section snippets

Neuronal cell cultures

SK–N–SH (KCLB 30011, human brain neuroblastoma) and U87-MG (KCLB 30014, human brain PTEN-null glioblastoma) cells were cultured in Eagle's minimum essential medium (MEM) with 2.0 mM l-glutamine and 1.0 mM sodium pyruvate supplemented with 10% heat-inactivated fetal bovine serum. Cells were grown to confluence at 37 °C in 5% CO2.

Cell viability assay

For mitochondrial tetrazolium assay (MTT) procedure, cells were seeded 1×104 cells/well in 96-well tissue culture plates. The confluent cells received MEM medium with 1%

Cell viability in SK–N–SH and U87-MG cells

SK–N–SH cells and U87-MG cells of sPTEN, but not wild-type U87-MG cells, showed PTEN expression in Western blot, whereas all three cell types showed maxi-K channel α-subunit and Akt expression, suggestive of PTEN-null glioblastoma cell line (Fig. 1A). The cell viability was concentration-dependently decreased in response to TNF-α (approximately 1–100 ng/ml) in the SK–N–SH cells and U87-MG cells with sPTEN (ANOVA, P<0.001), but not in the wild-type U87-MG cells. The viability of SK–N–SH cells

Discussion

In the present study, elevation of phosphorylated CK2 by KR-31378 was accompanied by decreased P-PTEN levels in association with increased P-Akt and P-CREB levels, and all these effects of KR-31378 were well antagonized by iberiotoxin, a maxi-K channel blocker. These findings have highlighted the hypothesis that the maxi-K channel opening-linked increase in P-CK2 and that suppression of P-PTEN lead to activation of Akt/CREB/Bcl-2 and repression of Bax production, thereby preventing the neuronal

Acknowledgments

We are grateful to Jonathan Kaskin for reading and commenting on the manuscript.

References (32)

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This study was supported, in part, by funds from the Critical National Technology Program of the Korea Science and Engineering Foundation (KWH, S-OK, S-EY).

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