Cytisine is neuroprotective in female but not male 6-hydroxydopamine lesioned parkinsonian mice and acts in combination with 17-ß-estradiol to inhibit apoptotic endoplasmic reticulum stress in dopaminergic neurons.

Apoptotic endoplasmic reticulum (ER) stress is a major mechanism for dopaminergic (DA) loss in Parkinson's disease (PD). We assessed if low doses of the partial α4ß2 nicotinic acetylcholine receptor agonist, cytisine attenuates apoptotic ER stress and exerts neuroprotection in substantia nigra pars compacta (SNc) DA neurons. Alternate day intraperitoneal injections of 0.2 mg/kg cytisine were administered to female and male mice with 6-hydroxydopamine (6-OHDA) lesions in the dorsolateral striatum, which caused unilateral degeneration of SNc DA neurons. Cytisine attenuated 6-OHDA-induced PD-related behaviors in female, but not in male mice. We also found significant reductions in tyrosine hydroxylase (TH) loss within the lesioned SNc of female, but not male mice. In contrast to female mice, DA neurons within the lesioned SNc of male mice showed a cytisine-induced pathological increase in the nuclear translocation of the pro-apoptotic ER stress protein, C/EBP homologous protein (CHOP). To assess the role of estrogen in cytisine neuroprotection in female mice, we exposed primary mouse DA cultures to either 10 nM 17-ß-estradiol and 200 nM cytisine or 10 nM 17-ß-estradiol alone. 17-ß-estradiol reduced expression of CHOP, whereas cytisine exposure reduced 6-OHDA-mediated nuclear translocation of two other ER stress proteins, activating transcription factor 6 and x-box-binding protein 1, but not CHOP. Taken together, these data show that cytisine and 17-ß-estradiol work in combination to inhibit all three arms (activating transcription factor 6, x-box-binding protein 1, and CHOP) of apoptotic ER stress signaling in DA neurons, which can explain the neuroprotective effect of low-dose cytisine in female mice.

Salubrinal attenuates nitric oxide mediated PERK:IRE1α: ATF-6 signaling and DNA damage in neuronal cells.

The present study was conducted to investigate the effect of salubrinal on nitric oxide mediated endoplasmic reticulum stress signaling and neuronal apoptosis. Rotenone treatment to neuro2a cells caused significantly decreased cell viability, increased cytotoxicity, augmented nitrite levels, increased nitrotyrosine level and augmented level of key ER stress markers (GRP-78, GADD153 and caspase-12). These augmented levels of ER stress markers could be attenuated with pretreatment of nitric oxide synthase inhibitor-aminoguanidine as well as with salubrinal. The rotenone treatment to neuro2a cells also triggered the ER stress induced up regulation of various signaling factors of unfolded protein response involving pPERK, ATF4, p-IRE1α, XBP-1 and ATF-6. Pretreatment of salubrinal significantly attenuated the activation of transmembrane kinases (PERK and IRE1) and ATF6 and restored the rotenone induced altered level of other UPR related signaling factors. Rotenone induced dephosphorylation of eIF2α was also inhibited with salubrinal treatment. Biochemically rotenone treatment to neuro2a cells caused the reactive oxygen species generation, depleted mitochondrial membrane potential and increased intra cellular calcium level which was attenuated with salubrinal treatment. Rotenone treatment to neuro2a cells also caused neuronal apoptosis, DNA fragmentation and chromatin condensation which were attenuated with salubrinal treatment. In conclusion, the findings suggested that rotenone causes the augmented level of nitric oxide which contributes in ER stress and could be inhibited by both aminoguanidine and/or salubrinal treatment. Further, salubrinal treatment attenuates the nitric oxide induced ER stress axis PERK:IRE1α:ATF-6 and inhibits the DNA damage and neuronal apoptosis.

The UPR Activator ATF6 Responds to Proteotoxic and Lipotoxic Stress by Distinct Mechanisms.

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.

Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells.

AIMS: Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. MAIN METHODS: The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. KEY FINDINGS: Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. SIGNIFICANCE: Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells.

Schisandrin B Ameliorates Myocardial Ischemia/Reperfusion Injury Through Attenuation of Endoplasmic Reticulum Stress-Induced Apoptosis.

Schisandrin B (Sch B), an active composition isolated from the fruit of Schisandra chinensis, has been proved to possess antiinflammatory, antioxidant and anti-endoplasmic reticulum (ER) stress effects in many rodent tissues. However, the exact mechanism of cardioprotective effect of Sch B still needs more study. Here, we detected the effects of Sch B on myocardial ischemia/reperfusion (I/R) injury rats. I/R injury model in this study was established by left anterior descending coronary artery ligation for 40 min followed by 1 h of reperfusion. Male healthy rats were randomly divided into five groups: the sham, I/R, Sch B (20 mg/kg) + I/R, and Sch B (40 mg/kg) + I/R, Sch B (80 mg/kg) + I/R, with 10 rats in each group. We showed that Sch B treatment significantly protected against myocardial I/R injury, as demonstrated by the decrease in the percentage of infarct formation assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining in representative heart tissue slices, comparing with the I/R control group. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), and total superoxide dismutase (T-SOD) were tested. The ER stress-related proteins such as C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6), and (PKR)-like ER kinase (PERK) were further measured by western blot, and their messenger RNA levels were measured by real-time PCR. The apoptosis of heart tissue cells was also tested through the expressions of caspase-9, caspase-3, Bcl-2, and Bax proteins. Collectively, these results revealed that Sch B exerts protection role on myocardial I/R injury through decreasing oxidative reaction, suppressing ATF6 and PERK pathway, and attenuating ER stress-induced apoptosis.

Endoplasmic reticulum stress increases brain MAPK signaling, inflammation and renin-angiotensin system activity and sympathetic nerve activity in heart failure.

We previously reported that endoplasmic reticulum (ER) stress is induced in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN) of heart failure (HF) rats and is reduced by inhibition of mitogen-activated protein kinase (MAPK) signaling. The present study further examined the relationship between brain MAPK signaling, ER stress, and sympathetic excitation in HF. Sham-operated (Sham) and HF rats received a 4-wk intracerebroventricular (ICV) infusion of vehicle (Veh) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10 µg/day). Lower mRNA levels of the ER stress biomarkers GRP78, ATF6, ATF4, and XBP-1s in the SFO and PVN of TUDCA-treated HF rats validated the efficacy of the TUDCA dose. The elevated levels of phosphorylated p44/42 and p38 MAPK in SFO and PVN of Veh-treated HF rats, compared with Sham rats, were significantly reduced in TUDCA-treated HF rats as shown by Western blot and immunofluorescent staining. Plasma norepinephrine levels were higher in Veh-treated HF rats, compared with Veh-treated Sham rats, and were significantly lower in the TUDCA-treated HF rats. TUDCA-treated HF rats also had lower mRNA levels for angiotensin converting enzyme, angiotensin II type 1 receptor, tumor necrosis factor-α, interleukin-1ß, cyclooxygenase-2, and NF-κB p65, and a higher mRNA level of IκB-α, in the SFO and PVN than Veh-treated HF rats. These data suggest that ER stress contributes to the augmented sympathetic activity in HF by inducing MAPK signaling, thereby promoting inflammation and renin-angiotensin system activity in key cardiovascular regulatory regions of the brain.

Advanced glycation end products upregulate the endoplasmic reticulum stress in human periodontal ligament cells.

BACKGROUND: The accumulation of advanced glycation end products (AGEs) appears to be the main factor responsible for modulating periodontal inflammation in diabetes. The aim of this study is to examine the effects of AGEs on inflammation in human periodontal ligament cells and to investigate the mechanism with a specific emphasis on the role of endoplasmic reticulum (ER) stress-induced nuclear factor-kappa B (NF-κB) pathway. METHODS: Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The protein expressions of ER markers and NF-κB were examined by Western blot analysis. The translocation of NF-κB was observed by immunofluorescence assay. Proinflammatory chemokine production was determined by enzyme-linked immunosorbent assay. RESULTS: Treatment with AGEs reduced cell viability in a concentration- and time-dependent manner. AGEs induced ER stress, as evidenced by survival molecules, such as glucose-regulated protein 78 (GRP78), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF-6), and apoptotic molecules, such as CCAAT/enhancer binding protein homologous protein (CHOP) and caspase 12. AGEs upregulated the nucleoprotein expression of NF-κB, enhanced translocation of NF-κB from the cytoplasm to the nucleus, and increased the production of proinflammatory chemokines interleukin-6 and interleukin-8. CONCLUSION: AGEs mediate inflammation of human periodontal ligament cells via the ER stress-induced NF-κB pathway.

Endoplasmic reticulum stress mediates house dust mite-induced airway epithelial apoptosis and fibrosis.

BACKGROUND: The endoplasmic reticulum (ER) stress response participates in many chronic inflammatory and autoimmune diseases. In the current study, we sought to examine the contribution of ER stress transducers in the pathogenesis of three principal facets of allergic asthma: inflammation, airway fibrosis, and airways hyperresponsiveness. METHODS: House Dust Mite (HDM) was used as an allergen for in vitro and in vivo challenge of primary human and murine airway epithelial cells. ER stress transducers were modulated using specific small interfering RNAs (siRNAs) in vivo. Inflammation, airway remodeling, and hyperresponsiveness were measured by total bronchoalveolar lavage (BAL) cell counts, determination of collagen, and methacholine responsiveness in mice, respectively. RESULTS: Challenge of human bronchiolar and nasal epithelial cells with HDM extract induced the ER stress transducer, activating transcription factor 6 α (ATF6α) as well as protein disulfide isomerase, ERp57, in association with activation of caspase-3. SiRNA-mediated knockdown of ATF6α and ERp57 during HDM administration in mice resulted in a decrease in components of HDM-induced ER stress, disulfide mediated oligomerization of Bak, and activation of caspase-3. Furthermore, siRNA-mediated knockdown of ATF6α and ERp57 led to decreased inflammation, airway hyperresponsiveness and airway fibrosis. CONCLUSION: Collectively, our work indicates that HDM induces ER stress in airway epithelial cells and that ATF6α and ERp57 play a significant role in the development of cardinal features of allergic airways disease. Inhibition of ER stress responses may provide a potential therapeutic avenue in chronic asthma and sub-epithelial fibrosis associated with loss of lung function.

Protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway plays a major role in reactive oxygen species (ROS)-mediated endoplasmic reticulum stress-induced apoptosis in diabetic cardiomyopathy.

BACKGROUND: Endoplasmic reticulum (ER) stress is considered one of the mechanisms contributing to reactive oxygen species (ROS)-mediated cell apoptosis. In diabetic cardiomyopathy (DCM), cell apoptosis is generally accepted as the etiological factor and closely related to cardiac ROS generation. ER stress is proposed the link between ROS and cell apoptosis; however, the signaling pathways and their roles in participating ER stress-induced apoptosis in DCM are still unclear. METHODS: In this study, we investigated the signaling transductions in ROS-dependent ER stress-induced cardiomocyte apoptosis in animal model of DCM. Moreover, in order to clarify the roles of IRE1 (inositol-requiring enzyme-1), PERK (protein kinase RNA (PKR)-like ER kinase) and ATF6 (activating transcription factor-6) in conducting apoptotic signal in ROS- dependent ER stress-induced cardiomocyte apoptosis, we further investigated apoptosis in high-glucose incubated cardiomyocytes with IRE1, ATF6 and PERK-knocked down respectively. RESULTS: we demonstrated that the ER stress sensors, referred as PERK, IRE1 and ATF6, were activated in ROS-mediated ER stress-induced cell apoptosis in rat model of DCM which was characterized by cardiac pump and electrical dysfunctions. The deletion of PERK in myocytes exhibited stronger protective effect against apoptosis induced by high-glucose incubation than deletion of ATF6 or IRE in the same myocytes. By subcellular fractionation, rather than ATF6 and IRE1, in primary cardiomyocytes, PERK was found a component of MAMs (mitochondria-associated endoplasmic reticulum membranes) which was the functional and physical contact site between ER and mitochondria. CONCLUSIONS: ROS-stimulated activation of PERK signaling pathway takes the major responsibility rather than IRE1 or ATF6 signaling pathways in ROS-medicated ER stress-induced myocyte apoptosis in DCM.

Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/reperfusion in rats: effect of neuroprotectant simvastatin.

Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examined the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

Tunicamycin sensitizes human melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by up-regulation of TRAIL-R2 via the unfolded protein response.

We have reported previously low expression of death receptors for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in fresh isolates and tissue sections of melanoma. This seemed to correlate with relative resistance of freshly isolated melanoma cells to TRAIL-induced apoptosis. We show in this study that the endoplasmic reticulum (ER) stress inducer, tunicamycin, selectively up-regulated the cell surface expression of TRAIL-R2, but not other members of the TNF receptor family, and enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. Tunicamycin-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was associated with increased activation of the caspase cascade and reduction in mitochondrial membrane potential and was inhibited by a recombinant TRAIL-R2/Fc chimeric protein. Up-regulation of TRAIL-R2 on the melanoma cell surface was associated with increased transcription of TRAIL-R2 and its total protein levels. Two signaling pathways of the ER stress-induced unfolded protein response mediated by inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha) and activation of transcription factor 6 (ATF6), respectively, seemed to be involved. In one melanoma line, there was clear evidence of activation of the IRE1alpha pathway, and small interfering RNA (siRNA) knockdown of IRE1alpha substantially reduced the up-regulation of TRAIL-R2. Similarly, there was evidence for the activation of the ATF6 pathway, and siRNA knockdown of ATF6 had a delayed effect on TRAIL-R2 expression in one but not another melanoma cell line. Moreover, the transcription factor CCAAT/enhancer-binding protein homologous protein seemed to be involved in the up-regulation of TRAIL-R2 by tunicamycin, but its role varied between different melanoma lines. Taken together, our results suggest that agents that induce ER stress may enhance TRAIL-R2 expression and increase the therapeutic response to TRAIL in melanoma.