Oh-Joo Kwon

Induction of unfolded protein response during neuronal induction of rat bone marrow stromal cells and mouse embryonic stem cells

When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2α phosphorylation. Transcription of two downstream targets of eIF2α, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.

TNFα-induced miR-130 resulted in adipocyte dysfunction during obesity-related inflammation

Adipocytes are continuously stimulated by proinflammatory cytokines such as TNFα, which cause adipocyte dysfunction by facilitating the inflammatory response. Although miR‐130 was reported to be an important regulator of adipogenesis by targeting PPARγ mRNA, little is known about the mechanisms regulating miR‐130 expression during the proinflammatory response. Here, we examined miR‐130 levels in white adipose tissue (WAT) from high‐fat diet (HFD) mice and TNFα‐stimulated adipocytes. Primary transcripts of miR‐130 were increased after TNFα stimulation, indicating that induction of miR‐130 during the pro‐inflammatory response is regulated by a transcriptional event. A chromatin immunoprecipitation assay showed that p65 binding to the promoter regions of miR‐130 was enhanced after TNFα treatment. Taken together, our findings suggest that induction of miR‐130 by TNFα is responsible for adipocyte dysfunction.

Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)‐depleted and control U2‐OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH‐depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP‐induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH‐depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD‐fmk, a pan‐specific caspase inhibitor. Large increases of LC3‐II were shown by immunoblot analysis of the SNP‐treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH‐depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP‐treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH‐depleted osteoblasts.

Alterations in mRNA Expression of Ribosomal Protein S9 in Hydrogen Peroxide-Treated Neurotumor Cells and in Rat Hippocampus After Transient Ischemia

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H2O2), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H2O2-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H2O2-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H2O2 treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H2O2. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.

X-box binding protein 1 enhances adipogenic differentiation of 3T3-L1 cells through the downregulation of Wnt10b expression

Differentiation of preadipocytes into adipocytes is controlled by various transcription factors. Recently, the pro‐adipogenic function of XBP1, a transcription factor upregulated by endoplasmic reticulum stress, has been reported. In this study, we demonstrated that XBP1 suppresses the expression of Wnt10b, an anti‐adipogenic Wnt, during the differentiation of 3T3‐L1 preadipocytes. The expression pattern of XBP1 was reciprocal to that of Wnt10b during the early stage of adipogenesis. The intracellular protein levels of β‐catenin were negatively regulated by XBP1. Direct binding of XBP1 to the Wnt10b promoter and the subsequent decrease of the β‐catenin signalling pathway represent a novel adipogenic differentiation mechanism.

Protective effects of vacuolar H+-ATPase c on hydrogen peroxide-induced cell death in C6 glioma cells

We have isolated a gene, the c subunit (ATP6L) of vacuolar H(+)-ATPase, involved in oxidative stress response. In this study, we examined the role of ATP6L and its molecular mechanisms in glial cell death induced by H(2)O(2). Expression of the ATP6L gene was increased by H(2)O(2) treatment in C6 glial cells. ATP6L siRNA-transfected C6 cells treated with H(2)O(2) showed a significant decrease in viability. ATP6L siRNA-transfected cells that were pretreated with MEK1/2 inhibitor completely recovered cell viability. Pretreatment of the transfected cells with zVAD-fmk, a pan-specific caspase inhibitor, did not result in the recovery of cell viability, as determined by a H(2)O(2)-induced cytotoxicity assay. The ultrastructural morphology of the transfected cells as seen by the use of transmission electron microscopy showed numerous cytoplasmic autophagic vacuoles with double membrane. These results suggest that ATP6L has a protective role against H(2)O(2)-induced cytotoxicity via an inhibition of the Erk1/2 signaling pathway, leading to inhibition of autophagic cell death.

Involvement of Sox-4 in the cytochrome c-dependent AIF-independent apoptotic pathway in HeLa cells induced by Delta12-prostaglandin J2.

Δ12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed Δ12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of Δ12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated Δ12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (Δ ψ m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the Δ ψ m was dissipated. One of the earliest events observed in Δ12-PGJ2-induced apoptotic events was dissipation of Δ ψ m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of Δ ψ m depolarization in Δ12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by Δ12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that Δ12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that Δ12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by Δ12-PGJ2.

X‐box binding protein 1 is a novel key regulator of peroxisome proliferator‐activated receptor γ2

X‐box binding protein 1 (XBP1), a transcription factor of the unfolded protein response, plays various roles in many biological processes. We examined its pro‐adipogenic activity and target genes during adipogenic differentiation in wild‐type and genetically modified 3T3‐L1 cells. Signalling pathways that contribute to Xbp1 mRNA splicing, and the correlation of the transcriptionally active XBP1 isoform (XBP1s) level with body mass index and the level of peroxisome proliferator‐activated receptor γ2 (PPARγ2) in human adipose tissues were also examined. The mRNA and nuclear protein expression levels of XBP1s increased immediately following hormonal induction of adipogenesis, reaching a peak at 6 h. Results from cDNA microarray and gene expression analyses using genetically modified cells indicated that PPARγ2 was a principal target of XBP1s. The XBP1s‐specific binding motif, which is distinct from the CCAAT/enhancer‐binding protein α binding site, was identified in the PPARγ2 promoter by site‐directed mutagenesis. Fetal bovine serum, insulin, 3‐isobutyl‐1‐methylxanthine and dexamethasone contributed independently to Xbp1 mRNA splicing. In human subcutaneous adipose tissues, the levels of both Xbp1s and Pparγ2 mRNA increased proportionally with body mass index, and there was a significant positive correlation between the two genes. These data suggest for the first time that positive regulation of PPARγ2 is a principal mechanism of XBP1s‐mediated adipogenesis in 3T3‐L1 cells.

Vacuolar H+ -ATPase c protects glial cell death induced by sodium nitroprusside under glutathione-depleted condition.

We examined the role of the c subunit (ATP6L) of vacuolar H+‐ATPase and its molecular mechanisms in glial cell death induced by sodium nitroprusside (SNP). ATP6L siRNA‐transfected cells treated with SNP showed a significant increase in cytotoxicity under glutathione (GSH)‐depleted conditions after pretreatment with buthionine sulfoximine, but reduction of ATP6L did not affect the regulation of lysosomal pH in analyses with lysosomal pH‐dependent fluorescence probes. Photodegraded SNP and ferrous sulfate induced cytotoxicity with the same pattern as that of SNP, but SNAP and potassium cyanide did not show activity. Pretreatment of the transfected cells with deferoxamine (DFO) reduced ROS production and significantly inhibited the cytotoxicity, which indicates that primarily iron rather than nitric oxide or cyanide from SNP contributes to cell death. Involvement of apoptotic processes in the cells was not shown. Pretreatment with JNK or p38 chemical inhibitor significantly inhibited the cytotoxicity, and we also confirmed that the MAPKs were activated in the cells by immunoblot analysis. Significant increase of LC3‐II conversion was observed in the cells, and the conversions were inhibited by cotransfection of the MAPK siRNAs and pretreatment with DFO. Introduction of Atg5 siRNA inhibited the cytotoxicity and inhibited the activation of MAPKs and the conversion of LC3. We finally confirmed autophagic cell death and involvement of MAPKs by observation of autophagic vacuoles via electron microscopy. These data suggest that ATP6L has a protective role against SNP‐induced autophagic cell death via inhibition of JNK and p38 in GSH‐depleted glial cells. J. Cell. Biochem. 112: 1985–1996, 2011.

Distribution of B/K protein in rat brain

Abstract. B/K protein is a recently isolated member of the double C2-like-domain protein family, which is highly abundant in rat brain. We generated high-titer rabbit polyclonal antibodies with specificity to the 55-kDa rat B/K protein, and examined the expression pattern of B/K protein in rat brain using an immunohistochemical staining method. Immunoreactivity to B/K protein was widely found in distinct regions of rat brain: strongly in the hypothalamus, most of the circumventricular organs, the locus coeruleus, the A5 neurons of the pons, and the anterior pituitary; moderately in the anterior olfactory nucleus, the raphe nucleus, the subfornical organ, and the median eminence; and faintly in the olfactory bulb, the telencephalon, the substantia nigra pars compacta, and the ventral tegmental area. In contrast, immunoreactivity to B/K protein was not observed in the thalamus, the cerebellum, the posterior pituitary, or the spinal cord. In most of the B/K-expressing neurons, immunoreactivity was expressed mainly in soma but not in nerve fibers. B/K was also expressed in nonneuronal cells such as the tanycytes and the subcommissural organ. In the vasopressin-secreting supraoptic and paraventricular nuclei of the hypothalamus, the site where B/K cDNA was originally isolated from, all of the neurons showing vasopressin immunoreactivity also expressed B/K protein, suggesting an overlap of their expression patterns.