Role of GS28 in sodium nitroprusside-induced cell death in cervical carcinoma cells.

Golgi S-nitro-N-acetylpenicillamine receptor complex 1 (GS28) has been implicated in Golgi vesicle transport. We examined the role of GS28 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using GS28 siRNA (siGS28)-transfected HeLa cells. Significant inhibition of cytotoxicity was observed in the cells treated with SNP, and photodegraded SNP showed equal cytotoxicity to SNP. Pretreatment with an ERK inhibitor or siErk1 cotransfection blocked the inhibition in cytotoxicity. Additionally, increased phosphorylation of ERK was maintained in the cells treated with SNP, and Nrf2 level was dependent on ERK phosphorylation. However, pretreatment with a pan-caspase inhibitor had no effect on cytotoxicity or procaspase-3 level. Pretreatment with an autophagy inhibitor or siATG5 cotransfection blocked the inhibition of cytotoxicity. The changes of LC3 corresponded to that in siErk1-cotransfected cells. These data suggest that GS28 has an inductive role in SNP-induced cell death via inhibition of ERK, leading to inhibition of autophagic processes in HeLa cells.

The IRE1α-XBP1s pathway promotes insulin-stimulated glucose uptake in adipocytes by increasing PPARγ activity.

The peroxisome proliferator-activated receptor-γ (PPARγ) improves whole-body insulin sensitivity by regulating the adipogenic and metabolic functions of mature adipocytes. We have previously demonstrated that an active splice variant of X-box binding protein 1 (XBP1s) enhances PPARγ expression during adipogenesis. In this study, we investigated the role of XBP1s, particularly with respect to PPARγ, in the mechanisms underlying insulin sensitivity in mature adipocytes. Insulin was able to stimulate XBP1s generation by activating inositol-requiring enzyme 1 (IRE1) α and was also able to increase its transcriptional activity by inducing nuclear translocation. XBP1s also upregulated the levels of phosphorylated IRS1 and AKT, demonstrating a positive feedback regulatory mechanism linking insulin and XBP1s. XBP1s enhanced the expression of fibroblast growth factor 21 and, in turn, increased PPARγ activity, translocation of GLUT4 to the cell surface, and glucose uptake rate in adipocytes. In addition, XBP1s abolished palmitate-induced insulin resistance in adipocytes by increasing adiponectin secretion, repressing the secretion of pro-inflammatory adipokines such as leptin, monocyte chemoattractant protein 1, and tumor necrosis factor α, and decreasing fatty acid release. These findings provide a novel mechanism by which XBP1s stimulate insulin sensitivity in adipocytes through fibroblast growth factor 21 induction and PPARγ activation.

Nuclear Expression of GS28 Protein: A Novel Biomarker that Predicts Prognosis in Colorectal Cancers.

Aims: GS28 (Golgi SNARE protein, 28 kDa), a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein family, plays a critical role in mammalian endoplasmic reticulum (ER)-Golgi or intra-Golgi vesicle transport. To date, few researches on the GS28 protein in human cancer tissues have been reported. In this study, we assessed the prognostic value of GS28 in patients with colorectal cancer (CRC). Methods and results: We screened for GS28 expression using immunohistochemistry in 230 surgical CRC specimens. The CRCs were right-sided and left-sided in 28.3% (65/230) and 71.3% (164/230) of patients, respectively. GS28 staining results were available in 214 cases. Among these, there were 26 nuclear predominant cases and 188 non-nuclear predominant cases. Stromal GS28 expression was noted in 152 cases of CRC. GS28 nuclear predominant immunoreactivity was significantly associated with advanced tumour stage (p = 0.045) and marginally associated with perineural invasion (p = 0.064). Decreased GS28 expression in the stromal cells was significantly associated with lymph node metastasis (N stage; p = 0.036). GS28 expression was not associated with epidermal growth factor receptor (EGFR) immunohistochemical positivity or KRAS mutation status. Investigation of the prognostic value of GS28 with Kaplan-Meier analysis revealed a correlation with overall survival (p = 0.004). Cases with GS28 nuclear predominant expression had significantly poorer overall survival than those with a non-nuclear predominant pattern. Conclusions: Taken together, these results indicate that GS28 nuclear predominant expression could serve as a prognostic marker for CRC and may help in identifying aggressive forms of CRC.

Oleate protects macrophages from palmitate-induced apoptosis through the downregulation of CD36 expression.

In obese patients, free fatty acids ectopically accumulated in non-adipose tissues cause cell death. Saturated fatty acids are more deleterious to non-adipose cells, and supplementation with monounsaturated fatty acids has been proposed to rescue cells from saturated fatty acid-induced cytotoxicity; however, the mechanisms are not well understood. To understand the cytoprotective role of monounsaturated fatty acids in lipotoxic cell death of macrophages, we investigated the antagonizing effect of oleate and the underlying mechanisms in palmitate-treated RAW264.7 cells. Palmitate strongly induced apoptosis in macrophages by increasing CD36 expression, which was identified to mediate both endoplasmic reticulum stress and the generation of reactive oxygen species. Co-treatment with oleate significantly reduced CD36 expression and its downstream signaling pathways of apoptosis in palmitate-treated cells. These findings provide a novel mechanism by which oleate protects macrophages from palmitate-induced lipotoxicity.

Aronia melanocarpa Extract Ameliorates Hepatic Lipid Metabolism through PPARγ2 Downregulation.

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Studies have demonstrated that anthocyanin-rich foods may improve hyperlipidemia and ameliorate hepatic steatosis. Here, effects of Aronia melanocarpa (AM), known to be rich of anthocyanins, on hepatic lipid metabolism and adipogenic genes were determined. AM was treated to C57BL/6N mice fed with high fat diet (HFD) or to FL83B cells treated with free fatty acid (FFA). Changes in levels of lipids, enzymes and hormones were observed, and expressions of adipogenic genes involved in hepatic lipid metabolism were detected by PCR, Western blotting and luciferase assay. In mice, AM significantly reduced the body and liver weight, lipid accumulation in the liver, and levels of biochemical markers such as fatty acid synthase, hepatic triglyceride and leptin. Serum transaminases, indicators for hepatocyte injury, were also suppressed, while superoxide dismutase activity and liver antioxidant capacity were significantly increased. In FL83B cells, AM significantly reduced FFA-induced lipid droplet accumulation. Protein synthesis of an adipogenic transcription factor, peroxisome proliferator-activated receptor γ2 (PPARγ2) was inhibited in vivo. Furthermore, transcriptional activity of PPARγ2 was down-regulated in vitro, and mRNA expression of PPARγ2 and its downstream target genes, adipocyte protein 2 and lipoprotein lipase were down-regulated by AM both in vitro and in vivo. These results show beneficial effects of AM against hepatic lipid accumulation through the inhibition of PPARγ2 expression along with improvements in body weight, liver functions, lipid profiles and antioxidant capacity suggesting the potential therapeutic efficacy of AM on NAFLD.

Long-term effects of duodenojejunal bypass on diabetes in Otsuka Long-Evans Tokushima Fatty rats.

BACKGROUND: Previous studies have shown that duodenojejunal bypass (DJB) resolves type 2 diabetes. However, this finding has been contradicted by several experimental and human trials and therefore needs to be clarified. METHODS: Otsuka Long-Evans Tokushima Fatty (OLETF) rats randomly underwent a sham operation or DJB. Thereafter, we measured daily body weight, serum levels of glucose and gut hormones such as glucagon-like peptide-1, insulin, and leptin. RESULTS: There was no significant difference in weight loss between rats in the DJB and sham-operated groups. There were also no differences in the area under the curve of glucose tolerance between the DJB and sham-operated groups (32466 ± 2261 mg/dL·min vs. 26319 ± 427 mg/dL·min; p = 0.35). Duodenojejunal bypass did not affect plasma concentrations of various gut hormones such as glucagon-like peptide-1, insulin, and leptin. CONCLUSIONS: We have shown that DJB alone does not improve glucose tolerance in obese, diabetic OLETF rats. Therefore, it may be that DJB alone is insufficient for diabetic control in obese diabetic rats. The addition of a restrictive component such as sleeve gastrectomy, or a new drug may be necessary for achieving diabetes reversal.

Nuclear Expression of GS28 Protein: A Novel Biomarker that Predicts Worse Prognosis in Cervical Cancers.

OBJECTIVE: The protein GS28 (28-kDa Golgi SNARE protein) has been described as a SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family member that plays a critical role in mammalian ER-Golgi or intra-Golgi vesicle transport. Little is known about the possible roles of GS28 in pathological conditions. The purpose of this study was to evaluate GS28 expression in cervical cancer tissues and explore its correlation with clinicopathological features and prognosis. METHODS: We investigated GS28 expression in 177 cervical cancer tissues by using immunohistochemistry and evaluated the correlation of GS28 expression with clinicopathological features, the expression of p53 and Bcl-2, and prognosis of cervical cancer patients. Immunoblotting was performed using six freshly frozen cervical cancer tissues to confirm the subcellular localization of GS28. RESULTS: Immunoreactivity of GS28 was observed in both nuclear and cytoplasmic compartments of cervical cancer cells. High nuclear expression of GS28 was associated with advanced tumor stages (P = 0.036) and negative expression of p53 (P = 0.036). In multivariate analyses, patients with high nuclear expression of GS28 showed significantly worse overall survival (OS) (hazard ratio = 3.785, P = 0.003) and progression-free survival (PFS) (hazard ratio = 3.019, P = 0.008), compared to those with low or no nuclear expression. It was also a reliable, independent prognostic marker in subgroups of patients with early stage T1 and negative lymph node metastasis in OS (P = 0.008 and 0.019, respectively). The nuclear expression of GS28 was confirmed by immunoblotting. CONCLUSION: High nuclear expression of GS28 is associated with poor prognosis in early-stage cervical cancer patients. GS28 might be a novel prognostic marker and a potential therapeutic target in cervical cancer treatment.

miR-148a is a downstream effector of X-box-binding protein 1 that silences Wnt10b during adipogenesis of 3T3-L1 cells.

Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3'UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and ß-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.

Raloxifene inhibits cloned Kv4.3 channels in an estrogen receptor-independent manner.

Raloxifene is widely used for the treatment and prevention of postmenopausal osteoporosis. We examined the effects of raloxifene on the Kv4.3 currents expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique and on the long-term modulation of Kv4.3 messenger RNA (mRNA) by real-time PCR analysis. Raloxifene decreased the Kv4.3 currents with an IC50 of 2.0 µM and accelerated the inactivation and activation kinetics in a concentration-dependent manner. The inhibitory effects of raloxifene on Kv4.3 were time-dependent: the association and dissociation rate constants for raloxifene were 9.5 µM(-1) s(-1) and 23.0 s(-1), respectively. The inhibition by raloxifene was voltage-dependent (δ = 0.13). Raloxifene shifted the steady-state inactivation curves in a hyperpolarizing direction and accelerated the closed-state inactivation of Kv4.3. Raloxifene slowed the time course of recovery from inactivation, thus producing a use-dependent inhibition of Kv4.3. ß-Estradiol and tamoxifen had little effect on Kv4.3. A preincubation of ICI 182,780, an estrogen receptor antagonist, for 1 h had no effect on the inhibitory effect of raloxifene on Kv4.3. The metabolites of raloxifene, raloxifene-4'-glucuronide and raloxifene-6'-glucuronide, had little or no effect on Kv4.3. Coexpression of KChIP2 subunits did not alter the drug potency and steady-state inactivation of Kv4.3 channels. Long-term exposure to raloxifene (24 h) significantly decreased the expression level of Kv4.3 mRNA. This effect was not abolished by the coincubation with ICI 182,780. Raloxifene inhibited Kv4.3 channels by interacting with their open state during depolarization and with the closed state at subthreshold potentials. This effect was not mediated via an estrogen receptor.

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.

Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state.

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1α stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

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 ß-catenin were negatively regulated by XBP1. Direct binding of XBP1 to the Wnt10b promoter and the subsequent decrease of the ß-catenin signalling pathway represent a novel adipogenic differentiation mechanism.

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.

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.

Inhibition of mouse brown adipocyte differentiation by second-generation antipsychotics.

Brown adipose tissue is specialized to burn lipids for thermogenesis and energy expenditure. Second-generation antipsychotics (SGA) are the most commonly used drugs for schizophrenia with several advantages over first-line drugs, however, it can cause clinically-significant weight gain. To reveal the involvement of brown adipocytes in SGA-induced weight gain, we compared the effect of clozapine, quetiapine, and ziprasidone, SGA with different propensities to induce weight gain, on the differentiation and the expression of brown fat-specific markers, lipogenic genes and adipokines in a mouse brown preadipocyte cell line. On Oil Red-O staining, the differentiation was inhibited almost completely by clozapine (40 µM) and partially by quetiapine (30 µM). Clozapine significantly down-regulated the brown adipogenesis markers PRDM16, C/EBPß, PPARγ2, UCP-1, PGC-1α, and Cidea in dose- and time-dependent manners, whereas quetiapine suppressed PRDM16, PPARγ 2, and UCP-1 much weakly than clozapine. Clozapine also significantly inhibited the mRNA expressions of lipogenic genes ACC, SCD1, GLUT4, aP2, and CD36 as well as adipokines such as resistin, leptin, and adiponectin. In contrast, quetiapine suppressed only resistin and leptin but not those of lipogenic genes and adiponectin. Ziprasidone (10 µM) did not alter the differentiation as well as the gene expression patterns. Our results suggest for the first time that the inhibition of brown adipogenesis may be a possible mechanism to explain weight gain induced by clozapine and quetiapine.

GS28 Protects Neuronal Cell Death Induced by Hydrogen Peroxide under Glutathione-Depleted Condition.

Golgi SNAP receptor complex 1 (GS28) has been implicated in vesicular transport between intra-Golgi networks and between endoplasmic reticulum (ER) and Golgi. Additional role(s) of GS28 within cells have not been well characterized. We observed decreased expression of GS28 in rat ischemic hippocampus. In this study, we examined the role of GS28 and its molecular mechanisms in neuronal (SK-N-SH) cell death induced by hydrogen peroxide (H(2)O(2)). GS28 siRNA-transfected cells treated with H(2)O(2) showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, which corresponded to an increase of intracellular reactive oxygen species (ROS) in the cells. Pretreatment of GS28 siRNA-transfected cells with p38 chemical inhibitor significantly inhibited cytotoxicity; we also observed that p38 was activated in the cells by immunoblot analysis. We confirmed the role of p38 MAPK in cotransfected cells with GS28 siRNA and p38 siRNA in the cell viability assay, flow cytometry, and immunoblot. Involvement of apoptotic or autophagic processes in the cells was not shown in the cell viability, flow cytometry, and immunoblot analyses. However, pretreatment of the cells with necrostatin-1 completely inhibited H(2)O(2)-induced cytotoxicity, ROS generation, and p38 activation, indicating that the cell death is necroptotic. Collectively these data imply that H(2)O(2) induces necroptotic cell death in the GS28 siRNA-transfected cells and that the necroptotic signals are mediated by sequential activations in RIP1/p38/ROS. Taken together, these results indicate that GS28 has a protective role in H(2)O(2)-induced necroptosis via inhibition of p38 MAPK in GSH-depleted neuronal 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.

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.

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 eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, 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.

Prolonged endoplasmic reticulum stress induces apoptotic cell death in an experimental model of chronic cyclosporine nephropathy.

BACKGROUND/AIMS: Apoptosis contributes to cyclosporine (CsA)-induced renal cell death. This study tested the effects of CsA-induced endoplasmic reticulum (ER) stress on apoptotic cell death in an experimental model of chronic CsA nephropathy. METHODS: CsA (15 mg/kg per day) was given to rats for 7 or 28 days. The ER stress response was evaluated with BiP expression, and the proapoptotic response was assessed with CHOP and caspase 12 expression. ER structure was evaluated by transmission electron microscopy, and apoptotic cell death was detected with TUNEL staining. RESULTS: Short-term treatment of CsA for 7 days activated both the ER stress response (induction of BiP mRNA and protein) and the proapoptotic response (upregulation of caspase 12 and CHOP mRNAs and proteins). However, long-term treatment with CsA for 28 days decreased BiP and further increased CHOP. The imbalance between the two responses coincided with the timing of the appearance of apoptotic cell death and the disruption of the ER structure. CONCLUSION: Prolonged CsA-induced ER stress causes apoptotic cell death by depleting molecular chaperones and activating the proapoptotic pathway.