Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development

Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.     

The Roles of the Ubiquitin–Proteasome System in the Endoplasmic Reticulum Stress Pathway

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells, which is essential for synthesis, processing, sorting of protein and lipid metabolism. However, the cells activate a defense mechanism called endoplasmic reticulum stress (ER stress) response and initiate unfolded protein response (UPR) as the unfolded proteins exceed the folding capacity of the ER due to the environmental influences or increased protein synthesis. ER stress can mediate many cellular processes, including autophagy, apoptosis and senescence. The ubiquitin-proteasome system (UPS) is involved in the degradation of more than 80% of proteins in the cells. Today, increasing numbers of studies have shown that the two important components of UPS, E3 ubiquitin ligases and deubiquitinases (DUBs), are tightly related to ER stress. In this review, we summarized the regulation of the E3 ubiquitin ligases and DUBs in ER stress.     

Lidocaine induces endoplasmic reticulum stress-associated apoptosis in vitro and in vivo

We demonstrated that upregulation of both gene expression of endoplasmic reticulum (ER) stress chaperones (BiP, calnexin, calreticulin, and PDI) and ER stress sensors (ATF6, IRE1 and PERK) was induced by lidocaine, a local anesthetic, in PC12 cells. In addition to gene regulation, lidocaine also induced typical ER stress phenomena such as ART6 proteolytic cleavage, eIF2 alpha phosphorylation, and XBP1 mRNA splicing. In in vivo experiments, while lidocaine downregulated gene expression of antiapoptotic factors (Bcl-2 and Bcl-xl), pro-apoptotic factor (Bak and Bax) gene expression was upregulated. Furthermore, lidocaine induced apoptosis, as measured histochemically, and upregulated PARP1, a DNA damage repair enzyme. These results are the first to show that lidocaine induces apoptosis through ER stress in vitro and in vivo.     

JI017, a Complex Herbal Medication,Induces Apoptosis via the Nox4–PERK–CHOP Axis in Ovarian Cancer Cells

Many anti-cancer drugs, including paclitaxel and etoposide, have originated and been developed from natural products, and traditional herbal medicines have fewer adverse effects and lesser toxicity than anti-tumor reagents. Therefore, we developed a novel complex herbal medicine, JI017, which mediates endoplasmic reticulum (ER) stress and apoptosis through the Nox4–PERK–CHOP signaling pathway in ovarian cancer cells. JI017 treatment increases the expression of GRP78, ATF4, and CHOP and the phosphorylation of PERK and eIF2α via the upregulation of Nox4. Furthermore, it increases the release of intracellular reactive oxygen species (ROS), the production of intracellular Ca²⁺, and the activation of exosomal GRP78 and cell lysate GRP78. Combination treatment using the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG) and JI017 reportedly induces increased ER stress and cell death in comparison to the control; however, knockdown experiments of PERK and CHOP indicated suppressed apoptosis and ER stress in JI017-treated ovarian cancer cells. Furthermore, targeting Nox4 using specific siRNA and pharmacological ROS inhibitors, including N-acetylcystein and diphenylene iodonium, blocked apoptosis and ER stress in JI017-treated ovarian cancer cells. In the radioresistant ovarian cancer model, when compared to JI017 alone, JI017 co-treatment with radiation induced greater cell death and resulted in overcoming radioresistance by inhibiting epithelial–mesenchymal-transition-related phenomena such as the reduction of E-cadherin and the increase of N-cadherin, vimentin, Slug, and Snail. These findings suggest that JI017 is a powerful anti-cancer drug for ovarian cancer treatment and that its combination treatment with radiation may be a novel therapeutic strategy for radioresistant ovarian cancer.     

Selective Activation of Endoplasmic Reticulum Stress by Reactive-Oxygen-Species-Mediated Ochratoxin A-Induced Apoptosis in Tubular Epithelial Cells

Ochratoxin A (OTA), one of the major food-borne mycotoxins, impacts the health of humans and livestock by contaminating food and feed. However, the underlying mechanism of OTA nephrotoxicity remains unknown. This study demonstrated that OTA induced apoptosis through selective endoplasmic reticulum (ER) stress activation in human renal proximal tubular cells (HK-2). OTA increased ER-stress-related JNK and precursor caspase-4 cleavage apoptotic pathways. Further study revealed that OTA increased reactive oxygen species (ROS) levels, and N-acetyl cysteine (NAC) could reduce OTA-induced JNK-related apoptosis and ROS levels in HK-2 cells. Our results demonstrate that OTA induced ER stress-related apoptosis through an ROS-mediated pathway. This study provides new evidence to clarify the mechanism of OTA-induced nephrotoxicity.     

Confounding Roles of ER Stress and the Unfolded Protein Response in Skeletal Muscle Atrophy

Skeletal muscle is an essential organ, responsible for many physiological functions such as breathing, locomotion, postural maintenance, thermoregulation, and metabolism. Interestingly, skeletal muscle is a highly plastic tissue, capable of adapting to anabolic and catabolic stimuli. Skeletal muscle contains a specialized smooth endoplasmic reticulum (ER), known as the sarcoplasmic reticulum, composed of an extensive network of tubules. In addition to the role of folding and trafficking proteins within the cell, this specialized organelle is responsible for the regulated release of calcium ions (Ca²⁺) into the cytoplasm to trigger a muscle contraction. Under various stimuli, such as exercise, hypoxia, imbalances in calcium levels, ER homeostasis is disturbed and the amount of misfolded and/or unfolded proteins accumulates in the ER. This accumulation of misfolded/unfolded protein causes ER stress and leads to the activation of the unfolded protein response (UPR). Interestingly, the role of the UPR in skeletal muscle has only just begun to be elucidated. Accumulating evidence suggests that ER stress and UPR markers are drastically induced in various catabolic stimuli including cachexia, denervation, nutrient deprivation, aging, and disease. Evidence indicates some of these molecules appear to be aiding the skeletal muscle in regaining homeostasis whereas others demonstrate the ability to drive the atrophy. Continued investigations into the individual molecules of this complex pathway are necessary to fully understand the mechanisms.     

Bax Inhibitor-1, a Conserved Cell Death Suppressor,Is a Key Molecular Switch Downstream from a Variety of Biotic and Abiotic Stress Signals in Plants

In Nature plants are constantly challenged by a variety of environmental stresses that could lead to disruptions in cellular homeostasis. Programmed cell death (PCD) is a fundamental cellular process that is often associated with defense responses to pathogens, during development and in response to abiotic stresses in fungi, animals and plants. Although there are many characteristics shared between different types of PCD events, it remains unknown whether a common mechanism drives various types of PCD in eukaryotes. One candidate regulator for such a mechanism is Bax Inhibitor-1 (BI-1), an evolutionary conserved, endoplasmic reticulum (ER)-resident protein that represents an ancient cell death regulator that potentially regulates PCD in all eukaryotes. Recent findings strongly suggested that BI-1 plays an important role in the conserved ER stress response pathway to modulate cell death induction in response to multiple types of cell death signals. As ER stress signaling pathways has been suggested to play important roles not only in the control of ER homeostasis but also in other biological processes such as the response to pathogens and abiotic stress in plants, BI-1 might function to control the convergence point that modulates the level of the “pro-survival and pro-death” signals under multiple stress conditions.     

Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Human Corneal Endothelial Cells from Endoplasmic Reticulum Stress-Mediated Apoptosis

Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy.     

Apoptosis Signal Regulating Kinase 1 (ASK1): Potential as a Therapeutic Target for Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia, characterized by a decline in memory and cognitive function. Clinical manifestations of AD are closely associated with the formation of senile plaques and neurofibrillary tangles, neuronal loss and cognitive decline. Apoptosis signal regulating kinase 1 (ASK1) is a mediator of the MAPK pathway, which regulates various cellular responses such as apoptosis, cell survival, and differentiation. Accumulating evidence indicates that ASK1 plays a key role in the pathogenesis of neurodegenerative disorders such as Huntington’s disease and AD. Of particular interest, ASK1 is associated with many signaling pathways, which include endoplasmic reticulum (ER) stress-mediated apoptosis, Aβ-induced neurotoxicity, tau protein phosphorylation, and insulin signal transduction. Here, we review experimental evidence that links ASK1 signaling and AD pathogenesis and propose that ASK1 might be a new point of therapeutic intervention to prevent or treat AD.     

Hyperthermia Induces Apoptosis through Endoplasmic Reticulum and Reactive Oxygen Species in Human Osteosarcoma Cells

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.     

Icariin Protects Rat Cardiac H9c2 Cells from Apoptosis by Inhibiting Endoplasmic Reticulum Stress

Endoplasmic reticulum stress (ERS) is one of the mechanisms of apoptotic cell death. Inhibiting the apoptosis induced by ERS may be a novel therapeutic target in cardiovascular diseases. Icariin, a flavonoid isolated from Epimedium brevicornum Maxim, has been demonstrated to have cardiovascular protective effects, but its effects on ERS are unknown. In the present study, we focused on icariin and investigated whether it might protect the cardiac cell from apoptosis via inhibition of ERS. In H9c2 rat cardiomyoblast cells, pretreatment of icariin significantly inhibited cell apoptosis by tunicamycin, an ERS inducer. Icariin also decreased generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential and activation of caspase-3. Moreover, icariin inhibited upregulation of endoplasmic reticulum markers, GRP78, GRP94 and CHOP, elicited by tunicamycin. These results indicated that icariin could protect H9c2 cardiomyoblast cells from ERS-mitochondrial apoptosis in vitro, the mechanisms may be associated with its inhibiting of GRP78, GRP94 and CHOP and decreasing ROS generation directly. It may be a potential agent for treating cardiovascular disease.     

The Inhibitory Effect of Quercetin on Asymmetric Dimethylarginine-Induced Apoptosis Is Mediated by the Endoplasmic Reticulum Stress Pathway in Glomerular Endothelial Cells

Asymmetric dimethylarginine (ADMA) is considered an independent mortality and cardiovascular risk factor in chronic kidney disease (CKD) patients, and contributes to the development of renal fibrosis. Quercetin (QC), a natural component of foods, protects against renal injury. Here, we explored the possible mechanisms that are responsible for ADMA-induced renal fibrosis and the protective effect of QC. We found that ADMA treatment activated the endoplasmic reticulum (ER) stress sensor proteins phosphorylated protein kinase RNA-activated-like ER kinase (PERK) and inositol requiring-1α (IRE1), which correspondingly induced C/EBP homologous protein (CHOP) expression and phosphorylated c-Jun N-terminal kinase (JNK) phosphorylation in glomerular endothelial cells (GEnCs). Following this, ADMA promoted ER stress-induced apoptosis and resulted in transforming growth factor β (TGF-β) expression in GEnCs. SP600125, an inhibitor of JNK, and CHOP siRNA protected against ADMA-induced cell apoptosis and TGF-β expression. QC prevented ADMA-induced PERK and IRE1 apoptotic ER stress pathway activation. Also, ADMA-induced GEnCs apoptosis and TGF-β expression was reduced by QC. Overexpression of CHOP blocked QC-mediated protection from apoptosis in ER stressed cells. Overall, these observations indicate that ADMA may induce GEnCs apoptosis and TGF-β expression by targeting the PERK-CHOP and IRE1-JNK pathway. In addition, drugs such as QC targeting ER stress may hold great promise for the development of novel therapies against ADMA-induced renal fibrosis.     

OSMI-1 Enhances TRAIL-Induced Apoptosis through ER Stress and NF-κB Signaling in Colon Cancer Cells

Levels of O-GlcNAc transferase (OGT) and hyper-O-GlcNAcylation expression levels are associated with cancer pathogenesis. This study aimed to find conditions that maximize the therapeutic effect of cancer and minimize tissue damage by combining an OGT inhibitor (OSMI-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found that OSMI-1 treatment in HCT116 human colon cancer cells has a potent synergistic effect on TRAIL-induced apoptosis signaling. Interestingly, OSMI-1 significantly increased TRAIL-mediated apoptosis by increasing the expression of the cell surface receptor DR5. ROS-induced endoplasmic reticulum (ER) stress by OSMI-1 not only upregulated CHOP-DR5 signaling but also activated Jun-N-terminal kinase (JNK), resulting in a decrease in Bcl2 and the release of cytochrome c from mitochondria. TRAIL induced the activation of NF-κB and played a role in resistance as an antiapoptotic factor. During this process, O-GlcNAcylation of IκB kinase (IKK) and IκBα degradation occurred, followed by translocation of p65 into the nucleus. However, combination treatment with OSMI-1 counteracted the effect of TRAIL-mediated NF-κB signaling, resulting in a more synergistic effect on apoptosis. Therefore, the combined treatment of OSMI-1 and TRAIL synergistically increased TRAIL-induced apoptosis through caspase-8 activation. Conclusively, OSMI-1 potentially sensitizes TRAIL-induced cell death in HCT116 cells through the blockade of NF-κB signaling and activation of apoptosis through ER stress response.     

Cellular Stress and Molecular Responses in Bladder Ischemia

The concept of bladder ischemia as a contributing factor to detrusor overactivity and lower urinary tract symptoms (LUTS) is evolving. Bladder ischemia as a consequence of pelvic arterial atherosclerosis was first documented in experimental models and later in elderly patients with LUTS. It was shown that early-stage moderate ischemia produces detrusor overactivity, while prolonged severe ischemia provokes changes consistent with detrusor underactivity. Recent studies imply a central role of cellular energy sensors, cellular stress sensors, and stress response molecules in bladder responses to ischemia. The cellular energy sensor adenosine monophosphate-activated protein kinase was shown to play a role in detrusor overactivity and neurodegeneration in bladder ischemia. The cellular stress sensors apoptosis signal-regulating kinase 1 and caspase-3 along with heat shock proteins were characterized as important contributing factors to smooth muscle structural modifications and apoptotic responses in bladder ischemia. Downstream pathways seem to involve hypoxia-inducible factor, transforming growth factor beta, vascular endothelial growth factor, and nerve growth factor. Molecular responses to bladder ischemia were associated with differential protein expression, the accumulation of non-coded amino acids, and post-translational modifications of contractile proteins and stress response molecules. Further insight into cellular stress responses in bladder ischemia may provide novel diagnostic and therapeutic targets against LUTS.     

Wogonin induces reactive oxygen species production and cell apoptosis in human glioma cancer cells

Glioma is the most common primary adult brain tumor with poor prognosis because of the ease of spreading tumor cells to other regions of the brain. Cell apoptosis is frequently targeted for developing anti-cancer drugs. In the present study, we have assessed wogonin, a flavonoid compound isolated from Scutellaria baicalensis Georgi, induced ROS generation, endoplasmic reticulum (ER) stress and cell apoptosis. Wogonin induced cell death in two different human glioma cells, such as U251 and U87 cells but not in human primary astrocytes (IC 50 > 100 μM). Wogonin-induced apoptotic cell death in glioma cells was measured by propidine iodine (PI) analysis, Tunnel assay and Annexin V staining methods. Furthermore, wogonin also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Moreover, treatment of wogonin also increased a number of signature ER stress markers glucose-regulated protein (GRP)-78, GRP-94, Calpain I, and phosphorylation of eukaryotic initiation factor-2α (eIF2α). Treatment of human glioma cells with wogonin was found to induce reactive oxygen species (ROS) generation. Wogonin induced ER stress-related protein expression and cell apoptosis was reduced by the ROS inhibitors apocynin and NAC (N-acetylcysteine). The present study provides evidence to support the fact that wogonin induces human glioma cell apoptosis mediated ROS generation, ER stress activation and cell apoptosis.