A Novel Rhynchophylline Analog, Y396, Inhibits Endothelial Dysfunction Induced by Oxidative Stress in Diabetes Through Epidermal Growth Factor Receptor.

Aims: Endothelial dysfunction appears in early diabetes mellitus partially because of epidermal growth factor receptor (EGFR) abnormal activation and downstream oxidative stress. The aim of this study was to determine whether Y396, a synthesized analog of rhynchophylline, could protect against endothelial dysfunction in diabetes and the underlying molecular mechanism. Results: Y396 could directly target the EGFR and inhibit its phosphorylation induced by high glucose and EGF, downstream translocation to the nucleus of E2F1, and its transcriptional activity and expression of Nox4. Diabetes-induced endothelium malfunction was ameliorated by Y396 treatment through EGFR inhibition. Downstream oxidative stress was decreased by Y396 in the aortas of type 1 diabetes mellitus mice and primary rat aorta endothelial cells (RAECs). Y396 could also ameliorate tunicamycin-induced oxidative stress in the aorta and RAECs. In addition, we again determined the protective effects of Y396 on high-fat diet/streptozotocin-induced type 2 diabetes mellitus. Innovation: This is the first study to demonstrate that Y396, a novel rhynchophylline analog, suppressed high-glucose-induced endothelial malfunction both in vivo and in vitro by inhibiting abnormal phosphorylation of EGFR. Our work uncovered EGFR as a novel therapeutic target and Y396 as a potential therapy against diabetes-induced complication. Conclusion: Y396 could directly bind with EGFR, and inhibit its phosphorylation and downstream E2F1 transcriptional activity. It could also preserve tunicamycin-evoked endothelial dysfunction and oxidative stress. It could protect against diabetes-induced endothelium malfunction in vivo through EGFR inhibition and downstream oxidative stress. Antioxid. Redox Signal. 32, 743-765.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells.

Insulin signaling pathway protects neuronal cell lines by Sirt3 mediated IRS2 activation.

Cellular stress like ER and oxidative stress are the principle causative agents of various proteinopathies. Multifunctional protein PARK7/DJ-1 provides protection against cellular stress. Recently, insulin/IGF also has emerged as a neuro-protective molecule. However, it is not known whether DJ-1 and insulin/IGF complement each other for cellular protection in response to stress. In this study, we show for the first time, that in human and mouse neuronal cell lines, down regulation of DJ-1 for 48 h leads to compensatory upregulation of insulin/IGF signaling (IIS) pathway genes, namely, insulin receptor, insulin receptor substrate, and Akt under normal physiological conditions as well as in cellular stress conditions. Moreover, upon exogenous supply of insulin there is a marked increase in the IIS components both at gene and protein levels leading to down regulation and inactivation of GSK3ß. By immunoprecipitation, it was observed that Sirt3 mediated deacetylation and activation of FoxO3a could not occur under DJ-1 downregulation. Transient DJ-1 downregulation also led to Akt mediated increased phosphorylation and nuclear exclusion of FoxO3a. When DJ-1 was downregulated increased interaction of Sirt3 with IRS2 was observed leading to its activation resulting in IIS upregulation. Thus, transient downregulation of DJ-1 leads to stimulation of IIS pathway by Sirt3 mediated IRS2 activation. Consequently, antiapoptotic program is triggered in neuronal cells via Akt-GSK3ß-FoxO3a axis. © 2018 BioFactors, 44(3):224-236, 2018.

Inhibition of endoplasmic reticulum stress and oxidative stress by vitamin D in endothelial cells.

Endoplasmic reticulum (ER) stress and oxidative stress promote endothelial dysfunction and atherosclerosis. Since vitamin D has been shown in several studies to lower the risk of cardiovascular disease, we examined the effects of vitamin D on ER stress and oxidative stress in endothelial cells. ER stress was measured using the placental secreted alkaline phosphatase assay and oxidative stress was measured by hydroethidine fluorescence. Expression of ER stress markers, including glucose-regulated protein 78, c-jun N-terminal kinase 1 phosphorylation, and eukaryotic initiation factor 2α phosphorylation, as well as X-box binding protein-1 splicing were measured in tunicamycin (TM)-treated human umbilical endothelial cells (HUVEC) treated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and other vitamin D analogs. When TM and 1,25-(OH)2D3 were added simultaneously, 1,25-(OH)2D3 prevented ER stress. However, the effect was much stronger when cells were pre-treated with 1,25-(OH)2D3 for 24-h. However, ER stress was not inhibited by 25-OH vitamin D3 (25-OHD3) or the vitamin D analog EB1089. Both ZK191784 and the vitamin D metabolite 24,25-dihydroxyvitamin D3 were as effective as 1,25-(OH)2D3 in preventing ER stress. Similar effects were observed dextrose-induced stress. All of the compounds tested, except for 25-OHD3, inhibited dextrose-induced (27.5mM) oxidative stress and ER stress. Although TM with and without 1,25-(OH)2D3 had no effect on VDR expression, inhibition of VDR expression via siRNA prevented 1,25-(OH)2D3, ZK191784, EB1089, and 24,25-dihydroxyvitamin D3 from inhibiting dextrose-mediated SO generation. Furthermore, each vitamin D analog, with the exception of 25-OHD3, prevented dextrose-induced toxicity. These results suggest that vitamin D has a protective effect on vascular endothelial cells.

Asymmetrical cross-talk between the endoplasmic reticulum stress and oxidative stress caused by dextrose.

AIMS: Oxidative and endoplasmic reticulum (ER) stresses are implicated in premature cardiovascular disease in people with diabetes. The aim of the present study was to characterize the nature of the interplay between the oxidative and ER stresses to facilitate the development of therapeutic agents that can ameliorate these stresses. MAIN METHODS: Human coronary artery endothelial cells were treated with varying concentrations of dextrose in the presence or absence of three antioxidants (alpha tocopherol, ascorbate and ebselen) and two ER stress modifiers (ERSMs) (4-phenylbutyrate and taurodeoxycholic acid). ER stress was measured using the placental alkaline phosphatase assay and superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence. KEY FINDINGS: The SO generation was increased with increasing concentrations of dextrose. The ER stress was increased with both low (0 and 2.75 mM) and high (13.75 and 27.5 mM) concentrations of dextrose. The antioxidants inhibited the dextrose induced SO production while in high concentrations they aggravated ER stress. The ERSM reduced ER stress and potentiated the efficacy of the three antioxidants. Tunicamycin-induced ER stress was not associated with increased SO generation. Time course experiments with a high concentration of dextrose or by overexpressing glucose transporter one in endothelial cells revealed that dextrose induced SO generation undergoes adaptive down regulation within 2 h while the ER stress is sustained throughout 72 h of observation. SIGNIFICANCE: The nature of the cross talk between oxidative stress and ER stress induced by dextrose may explain the failure of antioxidant therapy in reducing diabetes complications.

Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells.

The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide. Melatonin possesses various beneficial metabolic actions, decreased levels of which may accelerate T2D. Endoplasmic reticulum stress (ERS) has been linked to insulin resistance in multiple tissues, but the role of melatonin on ERS and insulin resistance in skeletal muscle has not yet been investigated. In this study, the results showed that tunicamycin decreased insulin-stimulated Akt phosphorylation, but promoted the phosphorylation of protein kinase R-like ER protein kinase (PERK) time-dependently in C2C12 cells. Consistently, ERS gene markers, including binding immunoglobulin protein (BIP)/glucose regulated protein 78 (GRP78) expression and the splicing of X box binding protein 1 (XBP-1), were activated by tunicamycin time-dependently. Interestingly, melatonin pretreatment reversed the elevated PERK phosphorylation, as well as the activation of Bip expression and XBP-1 splicing, and prevented the inhibitory effect of tunicamycin on Akt phosphorylation. In addition, the insulin-provoked glucose transport was reduced by tunicamycin, and then promoted by melatonin pretreatment. A strong phosphorylation of inositol-requiring enzyme 1 (IRE-1), c-JUN NH2-terminal kinase (JNK), and insulin receptor substrate 1 (IRS-1) serine, and simultaneously, a dramatic decrease of IRS-1 tyrosine phosphorylation were observed in the presence of tunicamycin, leading to a blockade of insulin signaling, which was reversed by melatonin pretreatment. Furthermore, luzindole pretreatment acted inversely with melatonin action on glucose uptake and insulin signaling. Therefore, these results demonstrated that melatonin pretreatment inhibited the activated role of tunicamycin on ERS and insulin resistance through melatonin receptor-mediated IRE-1/JNK/IRS-1 insulin signaling in skeletal muscle cells.

Inhibitory Effects of Verrucarin A on Tunicamycin-Induced ER Stress in FaO Rat Liver Cells.

Endoplasmic reticulum (ER) stress is linked with development and maintenance of cancer, and serves as a therapeutic target for treatment of cancer. Verrucarin A, isolated from the broth of Fusarium sp. F060190, showed potential inhibitory activity on tunicamycin-induced ER stress in FaO rat liver cells. In addition, the compound decreased tunicamycin-induced GRP78 promoter activity in a dose dependent manner without inducing significant inhibition of luciferase activity and cell growth for 6 and 12 h. Moreover, the compound decreased the expression of GRP78, CHOP, XBP-1, and suppressed XBP-1, and reduced phosphorylation of IRE1α in FaO rat liver cells. This evidence suggests for the first time that verrucarin A inhibited tunicamycin-induced ER stress in FaO rat liver cells.

Zonisamide suppresses endoplasmic reticulum stress-induced neuronal cell damage in vitro and in vivo.

Zonisamide has been reported to have protective effects on epilepsy and Parkinson׳s disease and to work via various mechanisms of action, such as inhibition of monoamine oxidase-B and enhancement of tyrosine hydroxylase. Recently, it has been suggested that zonisamide itself shows neuroprotective actions. Therefore, in the present study we investigated the neuroprotective effects of zonisamide against endoplasmic reticulum (ER) stress. We used human neuroblastoma (SH-SY5Y) cells and investigated the protective effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. In addition, we investigated the effect of zonisamide against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death and the mechanism of protection against ER stress. In vivo, we investigated the effect of zonisamide (20 mg/kg, p.o.) in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson׳s disease. Zonisamide not only suppressed MPP⁺-induced cell death, but also inhibited ER stress-induced cell death and suppressed the expression of ER stress-related factors such as C/EBO homologous protein (CHOP) in vivo. Furthermore, zonisamide inhibited the activation of caspase-3 in vitro. These results suggest that zonisamide affected ER stress via caspase-3. We think that ER stress, particularly the mechanism via caspase-3, is involved in part of the neuroprotective effect of zonisamide against the experimental models of Parkinson׳s disease.

Wogonin prevents rat dorsal root ganglion neurons death via inhibiting tunicamycin-induced ER stress in vitro.

Wogonin is a natural flavonoid isolated from the root of Scutellaria baicalensis Georgi, which has been widely used in various research areas for its anti-oxidant, anti-inflammatory, and anti-cancer activities. It also presents a neuroprotective effect in the brain while encounters stress conditions, but the mechanisms controlling the neuroprotective effect of wogonin are not clear. In this study, we investigated the biomechanism underlying the neuroprotective effect of wogonin on rat dorsal root ganglion (DRG) neurons. Wogonin pre-treatment at 75 µM significantly increased the cell viability of DRG neurons and decreased the number of the propidium iodide-positive DRG neurons before the endoplasmic reticulum (ER) stress is being induced by tunicamycin (TUN) (0.75 µg/mL). In addition, Wogonin also inhibited the release of LDH and up-regulated the level of GSH. Furthermore, wogonin decreased the activation of ER stress-related molecules, including glucose-regulated protein 78 (GRP78), GRP94, C/EBP-homologous protein, active caspase12 and active caspase3, phosphorylation of pancreatic ER stress kinase, and eukaryotic initiation factor 2 alpha (eIF2α). In summary, our results indicated that wogonin could protect DRG neurons against TUN-induced ER stress.

Identification of small molecules that protect pancreatic ß cells against endoplasmic reticulum stress-induced cell death.

Endoplasmic reticulum (ER) stress plays an important role in the decline in pancreatic ß cell function and mass observed in type 2 diabetes. Here, we developed a novel ß cell-based high-throughput screening assay to identify small molecules that protect ß cells against ER stress-induced cell death. Mouse ßTC6 cells were treated with the ER stressor tunicamycin to induce ER stress, and cell death was measured as a reduction in cellular ATP. A collection of 17600 compounds was screened for molecules that promote ß cell survival. Of the approximately 80 positive hits, two selected compounds were able to increase the survival of human primary ß cells and rodent ß cell lines subjected to ER stressors including palmitate, a free fatty acid of pathological relevance to diabetes. These compounds also restored ER stress-impaired glucose-stimulated insulin secretion responses. We show that the compounds promote ß cell survival by reducing the expression of key genes of the unfolded protein response and apoptosis, thus alleviating ER stress. Identification of small molecules that prevent ER stress-induced ß cell dysfunction and death may provide a new modality for the treatment of diabetes.

Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus.

Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to ß-lactam antibiotics. PBP2A cross-links nascent peptidoglycan when the native TPs are inhibited by ß-lactams. Although mecA expression is essential for ß-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes methicillin-resistant S. aureus (MRSA) strains to ß-lactams even though the ß-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and ß-lactams is noteworthy not simply because strategies to overcome MRSA are desperately needed but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The "synthetic lethality" of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic-resistant bacterial infections.

Endoplasmic reticulum stress induced by tunicamycin and antagonistic effect of Tiantai No.1 (1) on mesenchymal stem cells.

OBJECTIVE: Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum (ER). This ER function disorder is called endoplasmic reticulum stress (ERS). Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and so on. Therefore, the protection effect of the traditional Chinese drug-Tiantai No. 1 (1) on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. METHODS: Primarily cultured marrow mesenchymal stem cells (MSCs) of rats were treated by tunicamycin (TM) in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 (which would assist cells to resist cellular stress injury), and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. RESULTS: Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs (P<0.05). All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group (P<0.05 or P<0.01) after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group (P<0.05 or P<0.01). This effect showed a dose dependent manner. CONCLUSION: Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.

Tunicamycin-induced cell death in the trigeminal ganglion is suppressed by nerve growth factor in the mouse embryo.

The effect of nerve growth factor (NGF) on tunicamycin (Tm)-treated neurons in the trigeminal ganglion was investigated by use of caspase-3 immunohistochemistry. In intact embryos at embryonic day 16.5, only a few caspase-3-immunoreactivity were detected in the ganglion neurons. Mean +/- SE of the density of the immunoreactivity was 0.22 +/- 0.03%. In contrast, the number of the immunoreactive neurons was increased at 24 h after injection of 0.5 microg Tm in 1 microl of 0.05 N NaOH solution into mouse embryos at embryonic day 15.5. The density of immunoreactivity was also increased (mean +/- SE = 1.44 +/- 0.11%) compared to intact and 0.05 N NaOH-treated embryos (mean +/- SE = 0.35 +/- 0.03%). The Tm treatment caused increase of the number of trigeminal neurons representing apoptotic profiles (intact, mean +/- SE = 79.3 +/- 8.5; 0.05 N NaOH, mean +/- SE = 132 +/- 11.5; 0.5 microg Tm, mean +/- SE = 370.2 +/- 64.8). In addition, NGF significantly prevented the increase of density of the immunoreactivity (mean +/- SE = 0.54 +/- 0.16%) and the number of apoptotic cells (mean +/- SE = 146.2 +/- 11.3). Saline application (without NGF) had no effect on Tm-induced increase of the immunoreactivity (mean +/- SE = 1.78 +/- 0.23%) or the apoptotic profiles (mean +/- SE = 431.9 +/- 80.5). These results indicate that Tm-induced cell death in the trigeminal ganglion is suppressed by NGF in the mouse embryo.

Inhibition of endoplasm reticulum stress by ghrelin protects against ischemia/reperfusion injury in rat heart.

Ghrelin is a multi-functional polypeptide with cardiovascular protective effects. We aimed to explore whether the cardioprotective effect of ghrelin is mediated by inhibiting myocardial endoplasmic reticulum stress (ERS). A Langendorff model of isolated rat heart was used with ischemia/reperfusion (I/R; 40/120 min). Cardiac function was monitored, and histomorphologic features, degree of myocardial injury, level of ERS markers, and number of apoptotic cardiomyocytes were determined. Compared with control group, the I/R group showed significantly decreased cardiac function, seriously damaged myocardial tissue, increased number of apoptotic cells, and overexpression of mRNA and protein of ERS markers. However, preadministration of ghrelin in vivo (10(-8)mol/kg, intraperitoneal injection, every 12h, twice in all) greatly ameliorated the damaged heart function, attenuated myocardial injury and apoptosis, and decreased the expression of ERS markers: it decreased the mRNA and protein levels of glucose-regulated protein78 (GRP78) and C/EBP homologous protein (CHOP), with reduced caspase-12 protein expression. Furthermore, in vitro, ghrelin directly inhibited the myocardial ERS response induced by tunicamycin or dithiothreitol in rat cardiac tissue. Ghrelin could protect the heart against I/R injury, at least in part, through inhibiting myocardial ERS.

Structure of Deinococcus radiodurans tunicamycin-resistance protein (TmrD), a phosphotransferase.

The open-reading frame (ORF) DR_1419 in the Deinococcus radiodurans genome is annotated as a representative of the wide family of tunicamycin-resistance proteins as identified in a range of bacterial genomes. The D. radiodurans ORF DR_1419 was cloned and expressed; the protein TmrD was crystallized and its X-ray crystal structure was determined to 1.95 A resolution. The structure was determined using single-wavelength anomalous diffraction with selenomethionine-derivatized protein. The refined structure is the first to be reported for a member of the tunicamycin-resistance family. It reveals strong structural similarity to the family of nucleoside monophosphate kinases and to the chloramphenicol phosphotransferase of Streptomyces venezuelae, suggesting that the mode of action is possibly by phosphorylation of tunicamycin.

Nicotine suppresses tunicamycin-induced, but not thapsigargin-induced, expression of GRP78 during ER stress-mediated apoptosis in PC12 cells.

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.

Transforming growth factor-beta1 regulation of laminin gamma1 and fibronectin expression and survival of mouse mesangial cells.

The transforming growth factor-beta (TGF-beta) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-beta1 on laminin gamma1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-beta1 (10 ng/ml) stimulates laminin-gamma1 and fibronectin expression approximately two-fold in a time-dependent manner (0-48 h). TGF-beta1 treatment also retards laminin-gamma1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-beta1 increases the binding of laminin gamma1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin gamma1 is modified by N-linked glycosylation. TGF-beta1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin gamma1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-beta1 enhances mesangial cell viability and metabolic activities initially (0-24 h); however, eventually leads to cell death (24-48 h). TGF-beta1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-beta1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium.

Protective effect of nicotine on tunicamycin-induced apoptosis of PC12h cells.

Nicotine has been reported to have neuroprotective effects. The present study deals with the neuroprotective effect of nicotine on the tunicamycin-induced apoptosis of PC12h cells. Treatment of PC12h cells with tunicamycin causes endoplasmic reticulum stress leading to apoptosis. Nicotine dose-dependently prevented the tunicamycin-induced apoptosis. Hoechst 33258 staining demonstrated the protective effect of nicotine against tunicamycin-induced apoptosis. Treatment with nicotinic acetylcholine receptor (nAChR) and L-type voltage-sensitive calcium channel (L-VSCC) antagonists prevented the nicotine-induced protective effect. A phosphatidylinositol 3-kinase (PI3-K) inhibitor had no influence on the nicotine-induced neuroprotective effect. These results show that the neuroprotective effect of nicotine occurs through nAChRs including the alpha 7 subtype and L-VSCC in PC12h cells and not through the PI3-K/Akt pathway.

[Calcium signaling mediated by nicotine receptors in neurons].

Nicotine has many acute and chronic pharmacological effects. Nicotine treatment activates neuronal nicotinic acetylcholine receptors (nAChR) in peripheral and central nervous systems leading to depolarization and elevation of intracellular calcium levels, which are considered to cause stimulation of neurotransmitter release, synaptic transmission, intracellular signal transduction and gene expression. Multiple subtypes of nAChRs display different sensitivity to nicotinic agonists and antagonists. Each of these subtypes has a unique distribution in peripheral and central nervous systems. Although presynaptic nAChRs have been extensively studied to modulate the release of neurotransmitters, the functional importance of nAChRs in somata is not sufficiently characterized. To clarify the mechanisms of calcium signaling and its stimulation of gene expression via nAChRs in somata, we have investigated nAChR-mediating calcium signaling mechanisms including phosphorylation of p42/44 MAP kinase (ERK), CREB and Akt in PC12h cells. Nicotine transiently activates phosphorylation of ERK-, CREB and Akt. Nicotine induces the activation of both PI3 kinase/Act and ERK/CREB pathways via common pathways including non-alpha 7-nAChRs, L-type VSCC, CaM kinase and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt. Based on these results, we discuss nAChR signaling mechanisms in neurons.

Nerve growth factor attenuates endoplasmic reticulum stress-mediated apoptosis via suppression of caspase-12 activity.

Following endoplasmic reticulum (ER) stress, which occurs via inhibition of the glycosylation of newly synthesized proteins, caspase family proteins are activated to promote ER stress-mediated apoptosis. Here we report that nerve growth factor (NGF) suppressed the ER stress-mediated apoptosis in tunicamycin-treated PC12 cells through an extensive decrease of the caspase-3/-9/-12 activity. Detailed analysis of the mechanism underlying the NGF-mediated cell survival revealed that the activities of all seriate caspases were reduced through the phosphatidylinositol 3-kinase (PI3-K) signaling pathway induced by NGF. Moreover, we found that the activity of c-Jun N-terminal kinase (JNK) was not essential for the tunicamycin-induced apoptosis of PC12 cells. These results demonstrate that the inactivation of caspase-12 via the NGF-mediated PI3-K signaling pathway leads to inactivation of the caspase cascade including caspase-3 and -9.