Kentaro Oh-hashi

Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells.

Peroxynitrite, one of the most reactive radicals, is produced from superoxide anion and nitric oxide. A peroxynitrite generator, 3-morpholinosydonimine (SIN-1), was found to induce the expression of three different growth arrest and DNA damage-inducible (GADD) mRNA, GADD34, GADD45, and GADD153, at the early phase during cell death in human neuroblastoma SH-SY5Y cells. In addition, peroxynitrite activated p38 MAPK just before induction of three GADD mRNA. A specific inhibitor of p38 MAPK, SB202190, markedly suppressed peroxynitrite-induced expression of three GADD mRNA in SH-SY5Y cells. The expression of three GADD genes and also p38 MAPK phosphorylation were suppressed by treatment with radical scavengers, superoxide dismutase plus catalase and glutathione. Glutathione depletion by L-buthionine-S, R-sulfoximine (BSO), increased the vulnerability of the cells to peroxynitrite. These findings indicate that peroxynitrite-mediated oxidative stress activated p38 MAPK to induce three GADD genes.

Early growth responsive-1-dependent manganese superoxide dismutase gene transcription mediated by platelet-derived growth factor

Manganese superoxide dismutase (Mn‐SOD) plays a major role in protecting mitochondria from oxidative damage. Overexpression of Mn‐SOD maintains cell survival under conditions that lead to apoptotic death. In addition to the antioxidative enzyme, platelet‐derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation by activating survival signaling pathways in various cells. Here we show that PDGF induced the expression of the Mn‐SOD gene in NIH3T3 cells, and its induction was associated with early growth response‐1 (Egr‐1), a transcription factor. An electrophoretic mobility shift assay demonstrated that Egr‐1 bound to the proximal promoter of the Mn‐SOD gene in response to PDGF. The proximal promoter region of Mn‐SOD was shown to be transcriptionally responsive to both basal and PDGF stimulation by transfection studies. Forced expression of Egr‐1 in the cells activated Mn‐SOD transcription in a dose‐dependent manner. The pathway by which PDGF induced Egr‐1 involved the mitogen‐activated protein kinase kinase‐1 (MEK1) and extracellular signal‐regulated kinases 1 and 2 (ERK1/2), because the effect of PDGF on the induction of Egr‐1 was blocked by U0126, a specific MEK1 inhibitor. These findings indicate that the induction of Mn‐SOD is part of the anti‐apoptotic properties mediated by PDGF.

CRELD2 is a novel endoplasmic reticulum stress-inducible gene.

Recently, endoplasmic reticulum (ER) stress responses have been suggested to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we first identified cysteine-rich with EGF-like domains 2 (CRELD2) as an ER stress-inducible gene by analyzing a microarray analysis of thapsigargin (Tg)-inducible genes in Neuro2a cells. CRELD2 mRNA is also shown to be immediately induced by treatment with the ER stress-inducing reagents tunicamycin and brefeldin A. In the genomic sequence of the mouse CRELD2 promoter, we found a typical ER stress responsible element (ERSE), which is well conserved among various species. Using a luciferase reporter analyses, we demonstrated that the ERSE in mouse CRELD2 is functional and responds to Tg and ATF6-overexpression. Each mutation of ATF6- or NF-Y-binding sites in the ERSE of the mouse CRELD2 promoter dramatically decreased both the basal activity and responsiveness toward the ER stress stimuli. Our study suggests that CRELD2 could be a novel mediator in regulating the onset and progression of various ER stress-associated diseases.

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

J. Neurochem. (2011) 119, 839–847.

p44/42 MAP kinase and c-Jun N-terminal kinase contribute to the up-regulation of caspase-3 in manganese-induced apoptosis in PC12 cells

Caspase-3 (32 kDa) is one of the primary protease executioners of apoptosis and is activated by intra-chain proteolytic cleavage, which generates a large subunit (17 kDa) and a small subunit (12 kDa). Typically, after apoptotic stimuli, the level of cleaved caspase-3 increases while that of caspase-3 decreases. It has been shown that caspase-3 mRNA levels increase in cortex following traumatic brain injury or focal ischemia. In the present study, we demonstrate that both caspase-3 mRNA and protein increase in apoptotic PC12 cells following exposure to manganese which strongly induces caspase-3 cleavage. Up-regulation of caspase-3 protein was evident in manganese-treated PC12 cells and was moderate in cisplatin-, rotenone- and A23187-treated cells but was not observed in serum deprivation-, anisomycin-, camptothecin-, cycloheximide- or staurosporine-treated cells in which all treatments induced extensive DNA fragmentation. Manganese-induced up-regulation of caspase-3 mRNA was partially attenuated by the pretreatment with the MEK inhibitor U0126, but not with the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In contrast, the increase in caspase-3 protein was suppressed by both U0126 and SP600125. These results suggest that p44/42 MAPK contributes to the up-regulation of caspase-3 mRNA and the JNK pathway regulates caspase-3 protein levels posttranslationally in manganese-induced apoptosis in PC12 cells.

Amyloid-β peptides induce cell proliferation and macrophage colony-stimulating factor expression via the PI3-kinase/Akt pathway in cultured Ra2 microglial cells

Alzheimers disease is characterized by numerous amyloid‐β peptide (Aβ) plaques surrounded by microglia. Here we report that Aβ induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony‐stimulating factor (M‐CSF). We examined signal cascades for Aβ‐induced M‐CSF mRNA expression. The induction of M‐CSF was blocked by a phosphatidylinositol 3 kinase (PI3‐kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Aβ enhanced NF‐κB binding activity to the NF‐κB site of the mouse M‐CSF promoter, which was blocked by LY294002. These results indicate that Aβ induces M‐CSF mRNA expression via the PI3‐kinase/Akt/NF‐κB pathway.

NF-κB independent signaling pathway is responsible for LPS-induced GDNF gene expression in primary rat glial cultures

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta superfamily, was originally purified and cloned as a potent survival factor for midbrain dopaminergic neurons. Some studies have characterized the transcriptional regulation of the GDNF gene, but its regulatory mechanisms have yet to be well defined, especially under pathophysiological conditions. In this study, we used a pharmacological approach to study the expression of the rat GDNF gene induced by lipopolysaccharide (LPS) in primary cultures of glial cells. MG132, a blocker of nuclear factor kappaB (NF-kappaB) activation, did not apparently affect LPS-induced GDNF gene expression, whereas it attenuated the up-regulation of iNOS genes via Toll-like receptor (TLR) 4. In primary glial cultures, LPS increased the phosphorylation levels of c-Jun amino-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK); in primary microglial cultures, it enhanced phosphorylation of extracellular signal-regulated kinase (Erk). Of the several MAP kinase inhibitors tested, a JNK-specific inhibitor blocked LPS-induced GDNF transcription in primary cultures of microglia, but not of astrocytes. These results suggest that LPS up-regulates GDNF transcription through an NF-kappaB independent pathway, and that JNK is responsible for LPS-stimulated GDNF transcription in primary cultures of microglia.

Biosynthesis, processing, and secretion of glial cell line-derived neurotrophic factor in astroglial cells

Glial cell line-derived neurotrophic factor (GDNF) is synthesized as a precursor, proGDNF. However, the molecular mechanisms for the processing and secretion of GDNF are not fully characterized, since the amount of its biosynthesis and secretion in glial cells are below the detection limit of western blotting. We established stably GDNF-overexpressing C6 cells, and this enabled us to monitor its spontaneous secretion, as well as its processed forms in the cells. GDNF secretion was augmented by stimulation with high potassium, while it was inhibited by treatment with either tunicamycin, an inhibitor of protein glycosylation, or brefeldin A, a disturbing factor of ER-Golgi transport. Wild-type GDNF transfected cells secreted three forms of processed GDNF. After deglycosylation, the highest molecular weight of secreted GDNF showed the same mobility on electrophoresis as recombinant human GDNF without a whole pro-domain. Mutations in the pro-domain and two cysteines at the C-terminal of GDNF markedly diminished the secretion of resultant proteins into the culture medium. GDNF proteins having mutations in the putative furin-consensus sequence were secreted partly as unprocessed forms, and forms with lower molecular weights than a mature form were secreted from the C6 cells. Taking these observations together, we conclude that GDNF is likely secreted both with and without processing by furin-like proteases, and that the pro-domain and C-terminal cysteines of GDNF play important roles in its processing and secretion in cultured astrocytes and C6 cells.

ER calcium discharge stimulates GDNF gene expression through MAPK-dependent and -independent pathways in rat C6 glioblastoma cells

Glial cell line-derived neurotrophic factor (GDNF), a neurotrophic and differentiation factor, is expressed under several pathophysiological conditions but its regulatory signals have not yet been clarified. Here, we found that endoplasmic reticulum (ER) Ca(2+) discharge by thapsigargin induced GDNF mRNA as well as COX2 and GRP78 expression in rat C6 glioblastoma cells. GDNF mRNA was immediately induced and peaked at 2h by thapsigargin, and the alternative transcript consisting of exon 3 and exon 4 appeared to be most inducible. In spite of intracellular Ca(2+) perturbation, Ca(2+)-dependent PKC was not responsible for this induction. Instead, a PKCdelta-specific inhibitor, rottlerin, suppressed the thapsigargin-induced GDNF mRNA expression. On the other hand, thapsigargin transiently enhanced phosphorylation status of mitogen-activated protein kinase (MAPK) pathway, including extracellular signal-regulated kinase (Erk), p38 MAPK and c-JUN amino-terminal kinase1 (JNK1) simultaneously; whereas specific inhibitors against MEK1 and JNK only reduced the thapsigargin-induced GDNF mRNA expression. In addition, a pan-PKC inhibitor (Ro-31-8220) attenuated the thapsigargin-enhanced phosphorylation levels of Erk1/2 and JNK1, whereas rottlerin did not. Thus, the present study demonstrated that the thapsigargin-stimulated ER Ca(2+) discharge up-regulated GDNF gene expression through both MAPK-dependent and -independent pathways in C6 glioblastoma cells.

Biosynthesis and secretion of mouse cysteine-rich with EGF-like domains 2

In this study, we found that Cysteine‐rich with EGF‐like domains 2 (CRELD2), a novel endoplasmic reticulum stress‐inducible protein, is not only localized in the ER‐Golgi apparatus but also spontaneously secreted. Deletion of four C‐terminal amino acids from mouse CRELD2 or addition of tag‐peptides to its C‐terminus dramatically enhanced CRELD2 secretion. Intra‐ and extra‐cellular CRELD2 is differentially glycosylated and its spontaneous secretion was significantly prevented by overexpression of a dominant negative mutant Sar1 and treatment with brefeldin A. Overexpression of wild‐type GRP78 remarkably enhanced the secretion of wild‐type but not mutant CRELD2. Our results demonstrate both that CRELD2 is a novel secretory glycoprotein regulated by Sar1 and GRP78 and that the C‐terminal of CRELD2 plays a crucial role in its secretion.