Pilocarpine-induced status epilepticus causes N-methyl-D-aspartate receptor-dependent inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.     

Mechanism of reduced colonic contractility in experimental colitis: role of sarcoplasmic reticulum pump isoform-2

Inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis are inflammatory disorders associated with decreased colonic contractility. Here we show that, in experimental colitis in rat induced by trinitrobenzenesulfonic acid, there is a decrease in contraction in response to carbamoylcholine and the sarco/endoplasmic reticulum Ca⁺² (SERCA) pump inhibitor thapsigargin. However, the decrease in contractility may occur due to decrease in the SERCA pump levels or their inactivation. Therefore, we examined the protein and mRNA levels for SERCA2 isoform, which is predominant isoform in colonic smooth muscle. There was a decrease in the levels of SERCA2 protein and mRNA levels in inflamed colonic muscle. These findings suggest that decreased SERCA pump levels is responsible for a decrease in the Ca⁺² stores in the sarco/endoplasmic reticulum that causes a decrease in the contractility in colonic smooth muscle leading to poor bowel movements.     

Interaction sites among phospholamban, sarcolipin, and the sarco(endo)plasmic reticulum Ca(2+)-ATPase

A robust cross-link between Gln²³ in phospholamban (PLN) and Lys³²⁸ in the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA1a) is formed in the presence or absence of oxidant and is susceptible to both PLN phosphorylation and SERCA1a Ca²⁺ binding. This cross-link provides precisely the evidence needed to support our earlier proposal that collision of the PLN transmembrane helix at Asn²⁷ with the cytosolic extension of M4 at Leu³²¹ leads to unwinding of the helix. In a study of site-specific interactions among PLN, sarcolipin (SLN), and SERCA1a, we determined that mutations of some specific amino acids in PLN or SLN diminish either the super-inhibition imposed on SERCA1a function by the PLN-SLN binary complex or the physical interactions between PLN and SLN or both. These results have led to a revision of our earlier model for the PLN-SLN-SERCA1a complex.     

Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca²⁺-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca²⁺ homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca²⁺ concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.     

Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+‐transporters in heart failure

Zn²⁺‐homoeostasis including free Zn²⁺ ([Zn²⁺]_i) is regulated through Zn²⁺‐transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn²⁺‐transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, [Zn²⁺]_i was significantly high in doxorubicin‐treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKCα expression and PKCα‐phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in [Zn²⁺]_i using zinc‐ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn²⁺ transporters. Additionally, increased [Zn²⁺]_i could induce marked activation of PKCα. Moreover, we observed marked decrease in [Zn²⁺]_i under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn²⁺ transporters on an intersection pathway with increased [Zn²⁺]_i and PKCα activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well‐controlled [Zn²⁺]_i via Zn²⁺ transporters and PKCα can be important therapeutic approach in prevention/treatment of HF.     

Frequency-dependent contractile strength in mice over- and underexpressing the sarco(endo)plasmic reticulum calcium-ATPase

One of the prominent markers of end-stage heart failure at the molecular level is a decrease in function and/or expression of the sarcoplasmic reticulum ATPase protein [sarco(endo)plasmic reticulum calcium-ATPase, SERCA]. It has been often postulated that a decrease in SERCA pump activity can contribute in a major way to decreased cardiac function. To establish a functional relationship, we assessed how alterations in SERCA activity level affect basic contractile function in healthy myocardium devoid of other significant molecular changes. We investigated baseline contractile function, frequency-dependent activation, and beta-adrenergic response in ultrathin trabeculae isolated from hearts of mice overexpressing SERCA (transgenic, TG), underexpressing SERCA2a (heterozygous knockout, Het), and their respective wild-type (WT) littermates. At physiological temperature and frequency, compared with their respective WT littermates, SERCA1a mice displayed increased developed force at frequencies of 4-8 Hz ( approximately 90% increase at 4 Hz) and force equal to WT mice at 10-14 Hz. Force development at 4 Hz in presence of 1 muM isoproterenol was similar in TG and WT mice. In Het mice, developed force was nearly identical at the lower end of the frequency range (4-8 Hz) but slightly depressed at higher frequency (P < 0.05 at 14 Hz). In presence of 1 muM isoproterenol, developed force at 4 Hz was equal to that in WT mice. Compared with normal levels, increased SERCA activity enhanced force development only at subphysiological frequencies. A reduction in SERCA activity only showed a depression of force at the higher frequency range. Thus generalizations regarding the correlation between SERCA activity and contractility can be highly ambiguous, because this relationship is critically dependent on other factors including stimulation frequency.     

鼻病毒非结构蛋白2B 诱导细胞发生内质网应激

为探讨鼻病毒非结构蛋白2B诱导内质网应激和细胞凋亡的机制,本研究构建了鼻病毒非结构蛋白2B的真核表达载体p2B‐GFP ,通过转染BHK‐21细胞检测相关标志蛋白的变化情况。结果显示,非结构蛋白2B定位表达于BHK‐21细胞内质网,诱导内质网应激标志蛋白Grp78、CHOP的表达增加,并使活化转录因子6（ATF6）的转录活性增加,还诱导BHK‐21细胞发生核浓缩而凋亡,使凋亡标志蛋白PARP发生降解而减少。结果提示,鼻病毒非结构蛋白2B可诱导细胞发生内质网应激,并经该途径诱导细胞凋亡。     

SERP1 prevents hypoxia-reoxygenation-induced H9c2 apoptosis through activating JAK2/STAT3 pathway-dependent attenuation of endoplasmic reticulum stress

The endoplasmic reticulum (ER) stress plays an important role in myocardial ischemia/reperfusion (MI/R) injury. SERP1, the stress-associated endoplasmic reticulum protein 1, is involved in regulating ER stress response. However, whether it associates with MI/R injury is not identified. Here, we show that SERP1 is induced in the mouse heart after MI/R injury as well as in H9c2 cells under hypoxia/reoxygenation (H/R) treatment. Additionally, SERP1 overexpression reduces H/R-induced H9c2 apoptosis. Moreover, SERP1 overexpression suppresses H/R-induced ER stress and activates JAK2/STAT3 pathway. Furthermore, JAK2/STAT3 pathway inhibition by the specific inhibitor JSI-124 minimizes the suppressive effect of SERP1 overexpression on H/R-induced ER stress and H9c2 apoptosis. Together, these results uncover the protection of SERP1 against H/R-induced H9c2 apoptosis and further relate it to JAK2/STAT3 pathway-dependent attenuation of ER stress. This study suggests SERP1 as a potential regulator invovled in the pathophysiology of MI/R injury.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.     

Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.     

Hypoxia triggers endothelial endoplasmic reticulum stress and apoptosis via induction of VLDL receptor

Endothelial cells express very low density lipoprotein receptor (VLDLr). Beyond the function as peripheral lipoprotein receptor, other roles of VLDLr in endothelial cells have not been completely unraveled. In the present study, human umbilical vein endothelial cells were subjected to hypoxia, and VLDLr expression, endoplasmic reticulum (ER) stress, and apoptosis were assessed. Hypoxia triggered endothelial ER stress and apoptosis, and induced VLDLr expression. Silencing or stabilization of HIF-1α reduced and enhanced VLDLr expression, respectively. HIF-1α affected vldlr promoter activity by interacting with a hypoxia-responsive element (HRE). Knockdown or overexpression of VLDLr alleviated and exacerbated hypoxia-induced ER stress and apoptosis, respectively. Thus, hypoxia induces VLDLr expression through the interaction of HIF-1α with HRE at the vldlr promoter. VLDLr then mediates ER stress and apoptosis.     

Lipopolysaccharide prevents apoptosis induced by brefeldin A, an endoplasmic reticulum stress agent, in RAW 264.7 cells

The effect of lipopolysaccharide (LPS) on the cell death induced by endoplasmic reticulum (ER) stress agents in RAW 264.7 cells was studied. LPS prevented the cell death by brefeldin A, but not thapsigargin and tunicamycin. CpG DNA as well as LPS prevented brefeldin A-induced cell death whereas tumor necrosis factor-alpha or interferon-gamma did not. Brefeldin A-induced cell death was mediated with apoptotic cell death and it was significantly inhibited by LPS. LPS abolished the activation of ER stress-related caspases, such as caspases 1, 3, and 4. LPS prevented brefeldin A-induced morphological changes in RAW 264.7 cells. Further, LPS prevented brefeldin A-induced Golgi dispersion. Therefore, LPS was suggested to diminish the stress of ER/Golgi complexes induced by brefeldin A and inhibit apoptosis. The preventive action of LPS on brefeldin A-induced apoptosis is discussed.     

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to polydatin-induced apoptosis in human nasopharyngeal carcinoma CNE cells

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti-cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose- and time-dependent manner. The compound also dose-dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt in CNE cells, while knock-down of CCAAT/enhancer-binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti-tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin.     

Endoplasmic reticulum stress response is involved in the pathogenesis of stress induced gastric lesions in rats

Stress gastric ulcer is a serious complication, but the mechanism involved is not fully clarified. It is well known that mucosal cell apoptosis plays a crucial role in the pathogenesis of gastric ulceration. Recent studies have shown that endoplasmic reticulum (ER) stress is an important pathway leading to cellular apoptosis. To investigate the role of ER stress in the pathogenesis of stress gastric ulcer, we studied the alteration in the expression of ER stress markers GRP78 (glucose-regulated protein 78) and caspase-12 (an ER stress-specific proapoptotic molecule) and their relations with gastric mucosal apoptosis during development of stress gastric lesions in the water-immersion and restraint stress (WRS) model in rats. Rats developed severe gastric lesions after 6 h of WRS. Typical apoptosis was observed at the edge cells of WRS induced gastric lesions. Western blot analysis showed that GRP78 and activated caspase-12 were over-expressed in the gastric tissues of WRS rats. Immunohistochemical analysis demonstrated that increased GRP78 and caspase-12 were distributed only under the lesions. In addition, dithiothreitol and tunicamycin (ER stress inducers), which increased the expression of GRP78 and activated caspase-12, caused gastric mucosal injury and mucosal cell apoptosis in vitro. These findings suggest that ER stress might be involved in the development of stress gastric ulcer through an apoptotic mechanism.     

SelK is a novel ER stress-regulated protein and protects HepG2 cells from ER stress agent-induced apoptosis

Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis.     

Decreased sarcolipin protein expression and enhanced sarco(endo)plasmic reticulum Ca2+ uptake in human atrial fibrillation

Sarcolipin (SLN), a key regulator of cardiac sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase, is predominantly expressed in atria and mediates β-adrenergic responses. Studies have shown that SLN mRNA expression is decreased in human chronic atrial fibrillation (AF) and in aortic banded mouse atria; however, SLN protein expression in human atrial pathology and its role in atrial SR Ca(2+) uptake are not yet elucidated. In the present study, we determined the expression of major SR Ca(2+) handling proteins in atria of human AF patients and in human and in a mouse model of heart failure (HF). We found that the expression of SR Ca(2+) uptake and Ca(2+) release channel proteins are significantly decreased in atria but not in the ventricles of pressure-overload induced HF in mice. In human AF and HF, the expression of SLN protein was significantly decreased; whereas the expressions of other major SR Ca(2+) handling proteins were not altered. Further, we found that the SR Ca(2+) uptake was significantly increased in human AF. The selective downregulation of SLN and enhanced SR Ca(2+) uptake in human AF suggest that SLN downregulation could play an important role in abnormal intracellular Ca(2+) cycling in atrial pathology.     

Reticulon 3 mediates Bcl-2 accumulation in mitochondria in response to endoplasmic reticulum stress

Reticulon3 (RTN3), firstly isolated from the retina and widely expressed in human tissues with the highest expression in the brain, is presumed to play an important role in the developing axons through the transport of liquids and proteins. We have identified and characterized RTN3 as a RTN4B/ASY interaction protein. Here we demonstrated that ER-stress activated RTN3 expression. CHOP and ATF6 were sufficient to up-regulate the expression of RTN3. The down-regulation of RTN3 would induce apoptosis and attenuate the anti-apoptotic activity of Bcl-2, indicating RTN3 was required for the cellular survival and optimal anti-apoptotic activity of Bcl-2. Our present studies also indicated ER-stress induced RTN3 up-regulation could trigger Bcl-2 translocation from ER to mitochondria. Moreover, the previous studies showed that RTN4B was also a Bcl-2-interacted protein. We found that RTN3 and RTN4B could block the access of Bcl-2 to each other and thereafter determined the Bcl-2 subcellular distribution. Taken together, our findings indicate that RTN3 is directly involved in the ER-constituents trafficking events through dually acting as an essential and important ER-stress sensor, and a trigger for the Bcl-2 translocation. Q. Wan and E. Kuang contributed equally to this work.