78-kDa Glucose-regulated Protein Research Articles

Association of GRP78 promoter polymorphisms and serum GRP78 level with risk of asthenozoospermia.

The aim of this study was undertaken to investigate the association of 78-kDa glucose-regulated protein (GRP78) gene promoter polymorphisms with risk of asthenozoospermia (AZS) men. In addition, we performed association analysis between GRP78 promoter mutations and serum GRP78 level in asthenozoospermia. The study population comprised 400 subjects with AZS patients and 400 healthy controls. We assessed GRP78 rs3216733, rs17840761, and rs17840762 polymorphisms by using Snapshot SNP genotyping assays; serum GRP78 level was measured by enzyme-linked immunosorbent assay (ELISA). Semen quality was assessed by computer-assisted semen analysis. We found that rs3216733 was associated with increased risk of AZS (Gd vs. dd: adjusted OR = 1.42, 95% CI, 1.06-1.93, P= 0.020; Gd/GG vs. dd: adjusted OR = 1.43, 95% CI, 1.08-1.91, P= 0.013; G vs. d adjusted OR = 1.26, 95% CI, 1.03-1.56, P= 0.027). The haplotype analyses showed the frequency of G-C-C haplotype was significantly higher in AZS (P= 0.026). The percentage of progressive motility sperm was lower in the asthenozoospermic men with Gd and Gd/GG genotypes than dd genotype (P= 0.003). Moreover, the serum GRP78 levels were significantly lower in rs3216733 Gd/GG genotypes compared with the dd genotype (P< 0.001). Our findings suggest that rs3216733 Gd/GG genotypes contribute to poor sperm motility, probably by decreasing the level of GRP78.

TRPM7 channels play a role in high glucose–induced endoplasmic reticulum stress and neuronal cell apoptosis

High-glucose (HG) levels and hyperglycemia associated with diabetes are known to cause neuronal damage. The detailed molecular mechanisms, however, remain to be elucidated. Here, we investigated the role of transient receptor potential melastatin 7 (TRPM7) channels in HG-mediated endoplasmic reticulum stress (ERS) and injury of NS20Y neuronal cells. The cells were incubated in the absence or presence of HG for 48 h. We found that mRNA and protein levels of TRPM7 and of ERS-associated proteins, such as C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and inducible nitric-oxide synthase (iNOS), increased in HG-treated cells, along with significantly increased TRPM7-associated currents in these cells. Similar results were obtained in cerebral cortical tissue from an insulin-deficiency model of diabetic mice. Moreover, HG treatment of cells activated ERS-associated proapoptotic caspase activity and induced cellular injury. Interestingly, a NOS inhibitor, l-NAME, suppressed the HG-induced increase of TRPM7 expression and cellular injury. siRNA-mediated TRPM7 knockdown or chemical inhibition of TRPM7 activity also suppressed HG-induced ERS and decreased cleaved caspase-12/caspase-3 levels and cell injury. Of note, TRPM7 overexpression increased ERS and cell injury independently of its kinase activity. Taken together, our findings suggest that TRPM7 channel activities play a key role in HG-associated ERS and cytotoxicity through an apoptosis-inducing signaling cascade involving HG, iNOS, TRPM7, ERS proteins, and caspases.

APOL1 risk variants cause podocytes injury through enhancing endoplasmic reticulum stress.

Two coding sequence variants (G1 and G2) of Apolipoprotein L1 (APOL1) gene have been implicated as a higher risk factor for chronic kidney diseases (CKD) in African Americans when compared with European Americans. Previous studies have suggested that the APOL1 G1 and G2 variant proteins are more toxic to kidney cells than the wild-type APOL1 G0, but the underlying mechanisms are poorly understood. To determine whether endoplasmic reticulum (ER) stress contributes to podocyte toxicity, we generated human podocytes (HPs) that stably overexpressed APOL1 G0, G1, or G2 (Vec/HPs, G0/HPs, G1/HPs, and G2/HPs). Propidium iodide staining showed that HP overexpressing the APOL1 G1 or G2 variant exhibited a higher rate of necrosis when compared with those overexpressing the wild-type G0 counterpart. Consistently, the expression levels of nephrin and podocin proteins were significantly decreased in the G1- or G2-overexpressing cells despite the maintenance of their mRNA expressions levels. In contrast, the expression of the 78-kDa glucose-regulated protein ((GRP78), also known as the binding Ig protein, BiP) and the phosphorylation of the eukaryotic translation initiation factor 1 (eIF1) were significantly elevated in the G1/HPs and G2/HPs, suggesting a possible occurrence of ER stress in these cells. Furthermore, ER stress inhibitors not only restored nephrin protein expression, but also provided protection against necrosis in G1/HPs and G2/HPs, suggesting that APOL1 risk variants cause podocyte injury partly through enhancing ER stress.

New Hope for Pancreatic Ductal Adenocarcinoma Treatment Targeting Endoplasmic Reticulum Stress Response: A Systematic Review.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of tumours, and its incidence is rising worldwide. Although survival can be improved by surgical resection when these tumours are detected at an early stage, this cancer is usually asymptomatic, and disease only becomes apparent after metastasis. Several risk factors are associated with this disease, the most relevant being chronic pancreatitis, diabetes, tobacco and alcohol intake, cadmium, arsenic and lead exposure, certain infectious diseases, and the mutational status of some genes associated to a familial component. PDAC incidence has increased in recent decades, and there are few alternatives for chemotherapeutic treatment. Endoplasmic reticulum (ER) stress factors such as GRP78/BiP (78 kDa glucose-regulated protein), ATF6α (activating transcription factor 6 isoform α), IRE1α (inositol-requiring enzyme 1 isoform α), and PERK (protein kinase RNA-like endoplasmic reticulum kinase) activate the transcription of several genes involved in both survival and apoptosis. Some of these factors aid in inducing a non-proliferative state in cancer called dormancy. Modulation of endoplasmic reticulum stress could induce dormancy of tumour cells, thus prolonging patient survival. In this systematic review, we have compiled relevant results concerning those endoplasmic reticulum stress factors involved in PDAC, and we have analysed the mechanism of dormancy associated to endoplasmic reticulum stress and its potential use as a chemotherapeutic target against PDAC.

Rutin protects against lipopolysaccharide-induced mastitis by inhibiting the activation of the NF-κB signaling pathway and attenuating endoplasmic reticulum stress

Rutin, found widely in traditional Chinese medicine materials, is used to treat eye swelling and pain, hypertension, and hyperlipidemia. In the present study, a mouse mastitis model induced by lipopolysaccharide (LPS) was established to explore rutin's inhibitory mechanism on mastitis via nuclear factor kappa B (NF-κB) inflammatory signaling and the relationship between NF-κB signaling and endoplasmic reticulum (ER) stress. Mice were divided into six groups: Control group, LPS model group, LPS + rutin (25, 50, and 100mg/kg) and LPS + dexamethasone (DEX) group. DEX, rutin, and PBS (control and LPS groups) were administered 1h before and 12h after perfusion of LPS. After LPS stimulation for 24h, to evaluate rutin's therapeutic effect on mastitis, the mammary tissues of each group were collected to detect histopathological injury, tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 mRNA and protein levels; and glucose-regulated protein, 78kDa (GRP78) protein levels. The protein and mRNA levels of TNF-α, IL-1β, and IL-6 in the LPS + rutin group were significantly lower than those in the LPS model group. Similarly, p50/p105, phosphorylated (p)-p65/p65 and p-inhibitor of nuclear factor kappa b kinase subunit beta (p-IKKβ)/IKKβ ratios in the LPS + rutin group (50mg/kg) and LPS + rutin group (100mg/kg) decreased significantly. GRP78 protein expression was significantly higher in LPS + rutin group (100mg/kg). The structure of mammary tissue became gradually more intact and vacuolization of acini decreased as the rutin concentration increased. The nuclear quantity of p65 in the LPS + rutin group decreased significantly in a rutin dose-dependent manner. Rutin had an anti-inflammatory effect in the LPS-induced mouse mastitis model, manifested by inhibition of NF-κB pathway activation and attenuation of ER stress.

Estrogen receptor antagonist fulvestrant inhibits proliferation and promotes apoptosis of prolactinoma cells by regulating the IRE1/XBP1 signaling pathway.

The aim of the present study was to evaluate the effects of an estrogen receptor antagonist, fulvestrant, on proliferation and apoptosis of prolactinoma cells, and to reveal potential regulatory mechanisms. Prolactinoma GH3 cells were treated with 10‑6 mol/l fulvestrant for 2, 4, 8, 12 and 24h. GH3 cell growth was observed under a microscope and cell viability was detected by MTT assay. Morphological changes of the nuclei in GH3 cells were observed by Hoechst 33258 staining and apoptotic rates were detected by flow cytometry. Preprolactin (PPL) and prolactin (PRL) secretion levels from GH3 cells were measured using ELISA. In addition, the protein expression levels of inositol‑requiring enzyme1 (IRE1), X‑box binding protein (XBP)‑1 and glucose‑regulated protein, 78kDa (GRP78) in GH3 cells were detected by western blot analysis. Cell density and cell viability of GH3 cells were significantly reduced in a time‑dependent manner following treatment with fulvestrant (P<0.05). GH3 cells treated with fulvestrant also acquired an apoptotic morphology and the apoptotic rate of GH3 cells was significantly increased by fulvestrant in a time‑dependent manner (P<0.05). PPL and PRL secretion levels were significantly reduced by fulvestrant treatment in a time‑dependent manner (P<0.05). The protein expression levels of IRE1, XBP1 and GRP78 were also significantly reduced in a time‑dependent manner following treatment with fulvestrant (P<0.05). Therefore, fulvestrant may inhibit proliferation and promote apoptosis of GH3 cells by downregulating the IRE1/XBP1 signaling pathway.

The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells.

Dentin sialophosphoprotein (DSPP) is upregulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are commonly found under constant endoplasmic reticulum (ER) stress and exhibit increased levels of misfolded proteins, due to gene mutations and a stressful microenvironment. The present study examined the effects of DSPP silencing on the regulation of ER stress and the unfolded protein response (UPR) in oral cancer cells. A recently established stable DSPP short hairpin (sh)RNA-silenced OSC2 oral cancer cell line was used. The mRNA expression levels of ER stress-associated proteins, including 78kDa glucose-regulated protein (GRP78), sarcoplasmic/endoplasmic reticulum calcium ATPase 2b(SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3r), protein kinase R-like endoplasmic reticulum kinase(PERK), serine/threonine-protein kinase/endoribonuclease IRE1 (IRE1), activating transcription factor6 (ATF6) and matrix metalloproteinase20(MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B‑cell lymphoma2 (Bcl2), Bcl2-associated Xprotein(Bax) and cytochromec] and cell proliferation-related [proliferating cell nuclear antigen (PCNA)] proteins were analyzed by western blotting. Cell viability, apoptosis and migration were monitored by MTT assay, AnnexinV-fluorescein isothiocyanate flow cytometry and wound-healing assay, respectively. In transiently transfected puromycin‑free OSC2 cells, DSPP silencing markedly downregulated the mRNA expression levels of major ER stress regulators, including GRP78, SERCA2b, PERK, IRE1 and ATF6, as well as MMP20. DSPP silencing also resulted in decreased cell viability and migration, and enhanced apoptosis. Furthermore, PCNA and Bcl2 levels were decreased, whereas Bax and cytochromec protein levels were increased in DSPP-silenced OSC2 cells. Sustained puromycin treatment partially counteracted the effects of DSPP silencing on the mRNA expression levels of ER stress-related proteins and MMP20, and on the migratory capacity of OSC2 cells. However, following puromycin treatment of DSPP-silenced cells, cell viability was further reduced and apoptosis was enhanced. In conclusion, these data provide evidence to suggest that DSPP may be involved in ER stress mechanisms in oral squamous cell carcinoma, since its downregulation in OSC2 cells led to significant alterations in the levels of major ER stress-associated proteins, and subsequent collapse of the UPR system.

Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

Coronavirus tropism is predominantly determined by the interaction between coronavirus spikes and the host receptors. In this regard, coronaviruses have evolved a complicated receptor-recognition system through their spike proteins. Spikes from highly related coronaviruses can recognize distinct receptors, whereas spikes of distant coronaviruses can employ the same cell-surface molecule for entry. Moreover, coronavirus spikes can recognize a broad range of cell-surface molecules in addition to the receptors and thereby can augment coronavirus attachment or entry. The receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) is dipeptidyl peptidase 4 (DPP4). In this study, we identified membrane-associated 78-kDa glucose-regulated protein (GRP78) as an additional binding target of the MERS-CoV spike. Further analyses indicated that GRP78 could not independently render nonpermissive cells susceptible to MERS-CoV infection but could facilitate MERS-CoV entry into permissive cells by augmenting virus attachment. More importantly, by exploring potential interactions between GRP78 and spikes of other coronaviruses, we discovered that the highly conserved human GRP78 could interact with the spike protein of bat coronavirus HKU9 (bCoV-HKU9) and facilitate its attachment to the host cell surface. Taken together, our study has identified GRP78 as a host factor that can interact with the spike proteins of two Betacoronaviruses, the lineage C MERS-CoV and the lineage D bCoV-HKU9. The capacity of GRP78 to facilitate surface attachment of both a human coronavirus and a phylogenetically related bat coronavirus exemplifies the need for continuous surveillance of the evolution of animal coronaviruses to monitor their potential for human adaptations.

Lutein and Zeaxanthin Isomers Protect against Light-Induced Retinopathy via Decreasing Oxidative and Endoplasmic Reticulum Stress in BALB/cJ Mice.

Oxidative stress (OS) and endoplasmic reticulum stress (ERS) are the major factors underlying photoreceptor degeneration. Lutein, RR-zeaxanthin (3R,3’R-zeaxanthin) and RS (meso)-zeaxanthin (3R,3’S-RS- zeaxanthin) (L/Zi) could protect against cell damage by ameliorating OS in retina. In this study, we examined the effect of L/Zi supplementation in a mouse model of photoreceptor degeneration and investigated whether the treatment of L/Zi ameliorated OS and ERS. BALB/cJ mice after light exposure were used as the animal model. The protective effects of L/Zi were observed by electroretinography (ERG) and terminal deoxyuridine triphosphate nick-end labeling (TUNEL) analysis. The underlying mechanisms related to OS and ERS were explored by Western blotting. After L/Zi treatment, the ERG amplitudes were significantly higher, and the number of TUNEL-positive cells was significantly reduced compared to that of the vehicle group. Western blotting results revealed that OS was ameliorated according to the significant downregulation of phosphorylated c-Jun N-terminal kinase (p-JNK), and significant upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, ERS was reduced according to the significant downregulation of 78 kDa glucose-regulated protein (GRP78), phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), activating transcription factor 4 (ATF4) and activating transcription factor (ATF6). Our data shows that L/Zi provided functional and morphological preservation of photoreceptors against light damage, which is probably related to its mitigation of oxidative and endoplasmic reticulum stress.

Atorvastatin ameliorates early brain injury through inhibition of apoptosis and ER stress in a rat model of subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease with very poor prognosis. The aim of the present study was to evaluate the protective effects of atorvastatin on early brain injury (EBI) after SAH using a perforation SAH model. Male Sprague–Dawley rats were randomly divided into four groups: the sham group, the SAH group (model group), SAH + 10 mg.kg−1.day−1 atorvastatin (low atorvastatin group), and SAH + 20 mg.kg−1.day−1 atorvastatin (high atorvastatin group). Atorvastatin was administered orally by gastric gavage for 15 days before operation. At 24 h after SAH, we evaluated the effects of atorvastatin on brain water content, apoptosis by TUNEL assay and scanning electron microscope (SEM), and the expression of apoptosis-related proteins by immunofluorescence and Western blotting analysis. Compared with the sham group, we observed increased brain water content, significant apoptosis, and elevated levels of apoptosis-related proteins including caspase-3, CCAAT enhancer-binding protein homologous protein (CHOP), the 78-kDa glucose-regulated protein (GRP78), and aquaporin-4 (AQP4) in the SAH group. Atorvastatin administration under all doses could significantly reduce brain water content, apoptosis, and the expression levels of caspase-3, CHOP, GRP78, and AQP4 at 24 h after SAH. Our data show that early treatment with atorvastatin effectively ameliorates EBI after SAH through anti-apoptotic effects and the effects might be associated inhibition of caspase-3 and endoplasmic reticulum (ER) stress related proteins CHOP and GRP78.

The GRP94 gene of Yesso scallop (Patinopecten yessoensis): Characterization and expression regulation in response to thermal and bacterial stresses

The 94-kDa glucose-regulated protein (GRP94) belonging to the HSP90 family is an endoplasmic reticulum (ER) chaperone. It plays critical roles in ER quality control, and has been implicated as a specialized immune chaperone to regulate both innate and adaptive immunity. In this study, we identified and characterized a GRP94 gene (PyGRP94) from Yesso scallop (Patinopecten yessoensis). The protein sequence of PyGRP94 is highly conserved with its homologs in vertebrates, with a signal sequence in N-terminal, an ER retrieval signal sequence in C-terminal and a HATPase_c domain. Expression analysis suggests that PyGRP94 transcripts in early embryos are maternally derived and the zygotic expression is started from D-shaped larvae. This gene is also expressed in almost all the adult tissues examined except smooth muscle, with the highest expression level in hemocytes. Besides, PyGRP94 was demonstrated to be induced by heat shock and both Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacterial infection, with much more dramatic changes being observed after V. anguillarum challenge. Our results suggest the involvement of PyGRP94 in response to thermal stress, and that it might play an important role in the innate immune defense of scallop.

Protein tyrosine phosphatase 1B regulates endothelial endoplasmic reticulum stress; role in endothelial dysfunction

Protein tyrosine phosphatase 1B (PTP1B) impairs nitric oxide (NO) production and induces endothelial dysfunction in various diseases, including diabetes, septic shock and heart failure. In non-cardiovascular tissues, PTP1B modulates endoplasmic reticulum stress (ERS) however this role has never been assessed in endothelial cells. We evaluated the link between PTP1B, ERS and endothelial dysfunction in mice.Induction of ERS (Tunicamycin) in vivo in mice or ex vivo in mouse arteries led to severe arterial endothelial dysfunction (i.e. reduced flow-dependent, NO mediated dilatation in isolated small mesenteric arteries), and this was prevented by the PTP1B inhibitor trodusquemine and absent in PTP1B−/− mice. Trodusquemine also prevented the Tunicamycin -induced increased arterial levels of the molecular ERS actors 78 kDa glucose-regulated protein (GRP78) and Activating Transcription Factor 6 (ATF6α). Tunicamycin strongly increased the interactions of PTP1B with GRP78 and the activated forms of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and IRE1α (proximity Ligation Assay).Thus, PTP1B plays a central role in the regulation of ERS in the endothelium, and the endothelial protective effect of PTP1B inhibition appears likely due at least in part to reduction of endothelial ERS, notably by promoting PERK protective pathway. Modulation of ER stress via PTP1B inhibitors may be a promising approach to protect the endothelium in cardiovascular diseases.

Loss-of-function PCSK9 Mutants Evade the Unfolded Protein Response Sensor, GRP78, and Fail to Induce Endoplasmic Reticulum Stress when Retained

The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function (LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis was not observed. Furthermore, ER retention of endogenous PCSK9 via splice switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94-kDa glucose-regulated protein (GRP94) sequesters PCSK9 away from the 78-kDa glucose-regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78–PCSK9 complex and resulted in UPR activation following overexpression of ER-retained PCSK9 variants relative to WT secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9.

Tanshinone IIA exerts neuroprotective effects on hippocampus-dependent cognitive impairments in diabetic rats by attenuating ER stress-induced apoptosis

This study aimed to investigate the mechanism by which tanshinone IIA (Tan IIA) suppresses neuronal apoptosis in the hippocampus of diabetic rats. Sprague-Dawley (SD) rats were randomly divided into the following four groups: a control group, a diabetes group and diabetes groups treated with different doses (2 or 4 mg/kg/day) of Tan IIA. Streptozotocin (STZ) was injected into the rats to induce diabetes. Two days after STZ treatment, Tan IIA was intraperitoneally administered to rats in the Tan IIA groups, whereas an equal volume of saline was administered to rats in the control and diabetes groups. After 6 weeks, a one-trial object recognition task and the Morris water maze were applied. The diabetes group displayed notably decreased learning and memory abilities compared with the control group (P < 0.05). Tan IIA rescued hippocampus-dependent memory. Superoxide dismutase (SOD) activity was reduced, and reactive oxygen species (ROS) production, malondialdehyde (MDA) content, and 78-kDa glucose-regulated protein (Grp78), growth arrest and DNA damage-inducible gene 153 (CHOP/GAD153) and cleaved caspase-3 levels were increased in the hippocampus of diabetic rats compared with that of control rats, changes that were accompanied by an increase in neuronal apoptosis in diabetic rats compared with control rats (P < 0.01). However, Tan IIA reduced the MDA content and GRP78 and CHOP expression by inducing SOD activity. Tan IIA attenuated neuronal apoptosis and improved learning and memory by suppressing endoplasmic reticulum (ER) stress activation.

Advanced glycation end products-induced insulin resistance involves repression of skeletal muscle GLUT4 expression

Little is known about advanced glycation end products (AGEs) participation in glucose homeostasis, a process in which skeletal muscle glucose transporter GLUT4 (Scl2a4 gene) plays a key role. This study investigated (1) the in vivo and in vitro effects of AGEs on Slc2a4/GLUT4 expression in skeletal muscle of healthy rats, and (2) the potential involvement of endoplasmic reticulum and inflammatory stress in the observed regulations. For in vivo analysis, rats were treated with advanced glycated rat albumin (AGE-albumin) for 12 weeks; for in vitro analysis, soleus muscles from normal rats were incubated with bovine AGE-albumin for 2.5 to 7.5 hours. In vivo, AGE-albumin induced whole-body insulin resistance; decreased (~30%) Slc2a4 mRNA and GLUT4 protein content; and increased (~30%) the nuclear content of nuclear factor NF-kappa-B p50 subunit (NFKB1), and cellular content of 78 kDa glucose-regulated protein (GRP78). In vitro, incubation with AGE-albumin decreased (~50%) the Slc2a4/GLUT4 content; and increased cellular content of GRP78/94, phosphorylated-IKK-alpha/beta, nuclear content of NFKB1 and RELA, and the nuclear protein binding into Slc2a4 promoter NFKB-binding site. The data reveal that AGEs impair glucose homeostasis in non-diabetic states of increased AGEs concentration; an effect that involves activation of endoplasmic reticulum- and inflammatory-stress and repression of Slc2a4/GLUT4 expression.

Propofol elicits autophagy via endoplasmic reticulum stress and calcium exchange in C2C12 myoblast cell line.

In this study, we investigated the relationship between propofol and autophagy and examined whether this relationship depends on ER stress, production of ROS (reactive oxygen species), and disruption of calcium (Ca2+) homeostasis. To this end, we measured C2C12 cell apoptosis in vitro, along with Ca2+ levels; ROS production; and expression of proteins and genes associated with autophagy, Ca2+ homeostasis, and ER stress, including LC3 (microtubule-associate protein 1 light chain 3), p62, AMPK (adenosine 5’-monophosphate (AMP)-activated protein kinase), phosphorylated AMPK, mTOR (the mammalian target of rapamycin), phosphorylated mTOR, CHOP (C/BEP homologous protein), and Grp78/Bip (78 kDa glucose-regulated protein). We found that propofol treatment induced autophagy, ER stress, and Ca2+ release. The ratio of phosphorylated AMPK to AMPK increased, whereas the ratio of phosphorylated mTOR to mTOR decreased. Collectively, the data suggested that propofol induced autophagy in vitro through ER stress, resulting in elevated ROS and Ca2+. Additionally, co-administration of an ER stress inhibitor blunted the effect of propofol.

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats.

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.

Inhibition of endoplasmic reticulum stress-induced autophagy sensitizes melanoma cells to temozolomide treatment.

The incidence of malignant melanoma is increasing. The discovery of agents specifically targeting the mutated cascades has provided a good response for patients with oncogenic B-Raf proto-оncogene, serine/threonine kinase (BRAF). However, numerous studies continue to focus on novel methods of treatment to overcome acquired resistance to novel drugs. Recently, it has been revealed that inhibition of endoplasmic reticulum (ER) stress chaperon 78kDa glucose-regulated protein 78 (GRP78) leads to down-regulation of autophagy and increased sensitivity to temozolomide (TMZ) treatment. Melanoma cells have a different sensitivity to TMZ treatment, which corresponds to the basal autophagy level. In the present study, we demonstrated that downregulation of GRP78 mitigated chemoresistance to TMZ in three melanoma cell lines. We found that downregulation of GRP78 led to inhibition of autophagy, cell cycle arrest in the G0/G1 phase, and activation of caspase-7-induced apoptosis, and this was affected by the initial autophagy level. Moreover, inhibition of GRP78 mitigated the combined TMZ and chloroquine effect. Our data revealed that autophagy inhibition through downregulation of ER stress response could overcome resistance to TMZ treatment in melanoma cells with a high basal level of autophagy treatment, which makes this combination a potential potent antitumor treatment for metastatic melanoma.

The Effect of Bornyl cis-4-Hydroxycinnamate on Melanoma Cell Apoptosis Is Associated with Mitochondrial Dysfunction and Endoplasmic Reticulum Stress.

Bornyl cis-4-hydroxycinnamate, an active compound isolated from Piper betle stems, was investigated in terms of its effects on A2058 and A375 melanoma cell proliferation and protein expression in this study. We used flow cytometric analysis to examine the early stages of apoptosis induced by bornyl cis-4-hydroxycinnamate in the two melanoma cell lines and employed comparative proteomic analysis to investigate the effects of this compound on protein expression in A375 cells. Master maps generated by PDQuest software from two-dimensional electrophoresis (2-DE) analysis of A375 cells showed that the expression levels of 35 proteins were significantly altered, with 18 proteins upregulated and 17 downregulated. The proteomics study identified several proteins that are involved in mitochondrial dysfunction and endoplasmic reticulum stress (ER stress), in addition to apoptosis-associated proteins, including prohibitin, hypoxia-upregulated protein 1, stress 70 protein, 78 kDa glucose-regulated protein (GRP78), and protein deglycase DJ-1 (protein DJ-1) in melanoma cells exposed to bornyl cis-4-hydroxycinnamate. The treatment also resulted in a marked decline of the mitochondrial membrane potential, in cytochrome C release into the cytosol, in the activation of Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad), caspase-3, and caspase-9, and in the decreased expression of p-Bad, B-cell lymphoma 2 (Bcl-2), Bcl-xl, and induced myeloid leukemia cell differentiation protein-1 (Mcl-1), indicating that apoptosis induced by bornyl cis-4-hydroxycinnamate was mediated by the mitochondria through the caspase-dependent pathway. Also, salubrinal (an eukaryotic initiation factor 2α inhibitor; eIF2α inhibitor) was able to protect the cells from bornyl cis-4-hydroxycinnamate-induced apoptosis. Bornyl cis-4-hydroxycinnamate-related cell death also implied that the protein kinase R-like endoplasmic reticulum kinase (PERK)–eIF2α–ATF4–CHOP signal pathways was activated upon bornyl cis-4-hydroxycinnamate treatment. Altogether, our results support the conclusion that bornyl cis-4-hydroxycinnamate-induced apoptosis in melanoma cells is associated with mechanisms correlated with the activation of caspase cascades, mitochondrial dysfunction, and endoplasmic reticulum stress, and indicate that this molecule has the potential to be developed as a chemotherapeutic agent for human melanoma.

Quantitative proteomics and biochemical analyses reveal the role of endoplasmin in the regulation of the expression and secretion of A Disintegrin And Metalloproteinase 12

A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in multiple cancers such as breast and cervical cancers and its high expression reduces the overall patient survival rate. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently in cells are not clear. Here, we use affinity purification and mass spectrometry to identify the ADAM12S-interacting proteins. Spectral counting and MaxQuant label-free quantification reveal that ADAM12S but not ADAM12L specifically interacts with a subset of endoplasmic reticulum proteins, such as endoplasmin (GRP94), 78 kDa glucose-regulated protein (GRP78), and UDP-glucose:glycoprotein glucosyltransferase I (UGGT1), that regulate the folding and processing of secreted proteins. Further biochemical experiments validate the interaction between ADAM12S and several of its interacting proteins. Computational docking analysis demonstrates that GRP94 preferentially interacts with ADAM12S over ADAM12L. The data also suggest that both the protein expression level and the secretion of ADAM12S are regulated by GRP94 expression and knockdown. Our results reveal a link between these two proteins that are highly expressed in cancer cells. Furthermore, our studies define a new ADAM12S-specific regulator that may contribute to the cancer development. SignificanceA Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in many cancers such as lung, breast, and cervical cancers. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently are not completely understood. We use affinity purification and label-free quantitative proteomics to identify the ADAM12S-interacting proteins. Our results reveal that ADAM12S specifically interacts with a subset of endoplasmic reticulum proteins, including endoplasmin (GRP94), UDP-glucose:glycoprotein glucosyltransferase I (UGGT1), and neutral α-glucosidase AB (GANAB). Computer modeling reveals that ADAM12S interacts with the surface amino acids of GRP94 more strongly than ADAM12L. Biochemical experiments further reveal that GRP94 regulates both the protein level and the secretion of ADAM12S. Database mining finds that both GRP94 and ADAM12 are highly expressed in multiple cancers and their high expression is correlated with poor patient survival rate. Taken together, our work discovers a new upstream regulator for ADAM12S, which may contribute to its distinct functions in the regulation of the migration and invasion of cancer cells.