Manganese activates autophagy to alleviate endoplasmic reticulum stress–induced apoptosis via PERK pathway

Overexposure to manganese (Mn) is neurotoxic. Our previous research has demonstrated that the interaction of endoplasmic reticulum (ER) stress and autophagy participates in the early stage of Mn‐mediated neurotoxicity in mouse. However, the mechanisms of ER stress signalling pathways in the initiation of autophagy remain confused. In the current study, we first validated that ER stress–mediated cell apoptosis is accompanied by autophagy in SH‐SY5Y cells. Then, we found that inhibiting ER stress with 4‐phenylbutyrate (4‐PBA) decreased ER stress–related protein expression and reduced cell apoptosis, whereas blocking autophagy with 3‐methyladenine (3‐MA) increased cell apoptosis. These data indicate that protective autophagy was activated to alleviate ER stress–mediated apoptosis. Knockdown of the protein kinase RNA‐like ER kinase (PERK) gene inhibited Mn‐induced autophagy and weakened the interaction between ATF4 and the LC3 promoter. Our results reveal a novel molecular mechanism in which ER stress may regulate autophagy via the PERK/eIF2α/ATF4 signalling pathway. Additionally, Mn may activate protective autophagy to alleviate ER stress–mediated apoptosis via the PERK/eIF2α/ATF4 signalling pathway in SH‐SY5Y cells.     

A2a adenosine receptor agonist improves endoplasmic reticulum stress in MIN6 cell line through protein kinase A/ protein kinase B/ Cyclic adenosine monophosphate response element-binding protein/ and Growth Arrest And DNA-Damage-Inducible 34/ eukaryotic Initiation Factor 2α pathways

Endoplasmic reticulum (ER) stress is one of the main molecular events underlying pancreatic beta cell (PBC) failure, apoptosis, and a decrease in insulin secretion. Recent studies have highlighted the fundamental role of A2a adenosine receptor (A2aR) in potentiation of insulin secretion and proliferation of PBCs. However, possible protective effects of A2aR signaling against ER stress have not been elucidated yet. Thus, in the present study, we aimed to investigate the effects of A2aR activation in MIN6 beta cells undergoing tunicamycin (TM)-mediated ER stress. A2aR expression and activity were evaluated using real-time polymerase chain reaction and measurement of the cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), phospho-protein kinase B or Akt (p-Akt)/Akt, and phospho-Cyclic adenosine monophosphate response element-binding protein/CREB levels in response to a specific agonist (CGS 21680). Survival and proliferation in TM and CGS 21680 cotreated cells were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining, colony formation, and 5-bromo-2′-deoxyuridine (Brdu) assays. In addition, the effects of A2aR stimulation on insulin secretion were evaluated using the enzyme-linked immunosorbent assay. B-cell lymphoma 2 (Bcl-2), phospho-eukaryotic Initiation Factor 2α (p-eIF2α)/eIF2α, growth arrest and DNA-damage-inducible 34 (GADD34), X-box binding protein 1 (XBP-1), spliced X-box binding protein 1 (XBP-1s), immunoglobulin heavy-chain-binding protein (BIP), and CCAAT-enhancer-binding protein homologous protein (CHOP) levels were evaluated using western blotting. Our results showed a decrease in A2aR expression and p-Akt/Akt and p-CREB/CREB levels in TM-pretreated cells. We also mentioned that CGS 21680 effectively increased cell survival, proliferation, and insulin secretion in TM-treated cells. The antiapoptotic effects were possibly mediated through Bcl-2 upregulation. Our western blotting results indicated that A2aR effectively downregulated p-eIF2α/eIF2α, XBP-1, XBP-1s, BIP, and CHOP levels, whereas GADD34 was upregulated. Altogether, the present study revealed that A2aR signaling through PKA/Akt/CREB mediators alleviated TM cytotoxicity effects in MIN6 beta cells. Thus, the stimulation of this receptor was seen as a new approach to control ER stress in the PBC cells.     

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.     

CD437 induces apoptosis in ovarian adenocarcinoma cells via ER stress signaling

A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1α) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.     

The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress

Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.     

Endoplasmic reticulum stress is induced and modulated by enterovirus 71

Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER‐resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2α, but infection with UV‐inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2α was dependent on PKR: eIF2α phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR‐eIF2α pathway. Pulse‐chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus‐dependent, ATF6‐independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1‐mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71‐induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.     

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)‐null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk‐mediated phosphorylation of eIF2α, in Oca2‐null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34‐PP1α phosphatase complex. Gadd34‐complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2‐null melanocyte sensitivity to thapsigargin. Thus, Oca2‐null melanocytes adapt to acute ER stress by disruption of pro‐apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress.     

Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.     

Aberrant, differential and bidirectional regulation of the unfolded protein response towards cell survival by 3'-deoxyadenosine

The unfolded protein response (UPR) is involved in a diverse range of pathologies triggered by endoplasmic reticulum (ER) stress. Endeavor to seek selective regulators of the UPR is a promising challenge towards therapeutic intervention in ER stress-related disorders. In the present report, we describe aberrant, differential and bidirectional regulation of the UPR by 3'-deoxyadenosine (cordycepin) towards cell survival. 3'-Deoxyadenosine blocked ER stress-induced apoptosis via inhibiting the IRE1-JNK pro-apoptotic pathway. 3'-Deoxyadenosine also inhibited apoptosis through reinforcement of the pro-survival eIF2α signaling without affecting PERK activity. It was associated with depression of GADD34 that dephosphorylates eIF2α, and dephosphorylation of eIF2α by salubrinal mimicked the anti-apoptotic effect of 3'-deoxyadenosine. Unexpectedly, although 3'-deoxyadenosine caused activation of eIF2α, it inhibited downstream pro-apoptotic events including induction of ATF4 and expression of CHOP. Cooperation of adenosine transporter and A3 adenosine receptor, but not A1/A2 receptors, mediated the pluripotent effects of 3'-deoxyadenosine. In mice, ER stress caused activation of JNK, expression of CHOP and induction of apoptosis in renal tubules. The apoptosis was significantly attenuated by administration with 3'-deoxyadenosine, and it was correlated with blunted induction of JNK and CHOP in the kidney. These results disclosed atypical pro-survival regulation of the UPR by 3'-deoxyadenosine, which may be advantageous for the treatment of intractable, ER stress-related disorders.     

Chromium alleviates glucose intolerance, insulin resistance, and hepatic ER stress in obese mice

Objective: Chromium has gained popularity as a nutritional supplement for diabetic patients. This study evaluated the effect of chronic administration of a chromium complex of d ‐phenylalanine (Cr(d ‐phe)₃) on glucose and insulin tolerance in obese mice. The study tested the hypothesis that Cr(d ‐phe)₃ suppresses endoplasmic reticulum (ER) stress and insulin resistance in these animals. Methods and Procedures: C57BL lean and ob/ob obese mice were randomly divided to orally receive vehicle or Cr(d ‐phe)₃ (3.8 μg of elemental chromium/kg/day) for 6 months. Insulin sensitivity was evaluated by glucose and insulin tolerance tests. Protein levels of phosphorylated pancreatic ER kinase (PERK), α subunit of translation initiation factor 2 (eIF2α) and inositol‐requiring enzyme‐1 (IRE‐1), p‐c‐Jun, and insulin receptor substrate‐1 (IRS‐1) phosphoserine‐307 were assessed by western blotting. In vitro ER stress was induced by treating cultured muscle cells with thapsigargin in the presence or absence of Cr(d ‐phe)₃. Results: ob/ob mice showed poor glucose and insulin tolerance compared to the lean controls, which was attenuated by Cr(d ‐phe)₃. Markers of insulin resistance (phospho‐c‐Jun and IRS‐1 phosphoserine) and ER stress (p‐PERK, p‐IRE‐1, p‐eIF2α), which were elevated in ob/ob mice, were attenuated following Cr(d ‐phe)₃ treatment. Chromium treatment was also associated with a reduction in liver triglyceride levels and lipid accumulation. In cultured myotubes, Cr(d ‐phe)₃ attenuated ER stress induced by thapsigargin. Discussion: Oral Cr(d ‐phe)₃ treatment reduces glucose intolerance, insulin resistance, and hepatic ER stress in obese, insulin‐resistant mice.