Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival

Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). We found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival.     

Effects of the Sigma-1 Receptor Agonist 1-(3,4-Dimethoxyphenethyl)-4-(3-Phenylpropyl)-Piperazine Dihydro-Chloride on Inflammation after Stroke

Activation of the sigma-1 receptor (Sig-1R) improves functional recovery in models of experimental stroke and is known to modulate microglia function. The present study was conducted to investigate if Sig-1R activation after experimental stroke affects mediators of the inflammatory response in the ischemic hemisphere. Male Wistar rats were subjected to transient occlusion of the middle cerebral artery (MCAO) and injected with the specific Sig-1R agonist 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (SA4503) or saline for 5 days starting on day 2 after MCAO. Treatment did not affect the increased levels of the pro-inflammatory cytokines interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13) in the infarct core and peri-infarct area after MCAO. In addition, treatment with SA4503 did not affect elevated levels of nitrite, TNF-α and IL-1β observed in primary cultures of microglia exposed to combined Hypoxia/Aglycemia, while the unspecific sigma receptor ligand 1,3-di-o-tolylguanidine (DTG) significantly decreased the production of nitrite and levels of TNF-α. Analysis of the ischemic hemisphere also revealed increased levels of ionized calcium binding adaptor molecule 1 (Iba1) levels in the infarct core of SA4503 treated animals. However, no difference in Iba1 immunoreactivity was detected in the infarct core. Also, levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and OX-42 were not increased in the infarct core in rats treated with SA4503. Together, our results suggest that sigma-1 receptor activation affects Iba1 expression in microglia/macrophages of the ischemic hemisphere after experimental stroke but does not affect post-stroke inflammatory mediators.     

Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor

We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell''s responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress.Sigma-1 receptor (Sig-1R) is expressed on endoplasmic reticulum (ER) membranes. Several functions have been attributed to Sig-1R, including regulation of ion channels such as Ca2+ and K+ channels, inhibition of Ca2+ influx through the N-methyl-D-aspartate (NMDA) receptor, modulation of the release of neurotransmitters such as dopamine, regulation of lipid distribution, cell differentiation, and behavioral sensitization to cocaine and other stimulants.¹ Recently, Sig-1R was shown to have neuroprotective activity, and several studies have demonstrated that it acts as a molecular chaperone.^{2, 3, 4} Under normal conditions, Sig-1R forms a complex with another molecular chaperone, GRP78/BiP, on the ER membrane. Under ER stress, Sig-1R dissociates from BiP, interacts with IP3 receptors, and stabilizes IP3 receptor structure.⁴Numerous studies have examined the role of Sig-1R in the pathogenesis of psychiatric diseases. Postmortem analysis has shown that Sig-1R expression is reduced in the brains of schizophrenia patients.⁵ Additionally, Sig-1R knockout mice exhibit symptoms of depression.⁶ Given these observations, it is possible that reduction of Sig-1R is a pathogenic factor in disorders such as schizophrenia and depression. Therefore, numerous synthetic compounds that bind to Sig-1R, including antidepressants and antipsychotic drugs, have been examined as therapeutic targets for these disorders.⁷ However, the results of clinical testing have not been satisfactory.⁸ One possible reason is that the effects of compounds that bind to Sig-1R cannot fully manifest because Sig-1R expression is reduced in the brains of patients with psychiatric diseases. This led us to examine whether compounds capable of inducing Sig-1R expression might be therapeutic in these diseases.When cells encounter ER stress, Sig-1R expression increases in response to activation of the PERK pathway, which is one of the cellular responses to ER stress.⁹ In addition, the induction of Sig-1R expression can repress cell death signals that accompany ER stress.⁹Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) that is widely used in clinical practice as an antidepressant. Because Flv is a potent Sig-1R agonist that exhibits a stronger affinity for Sig-1R than for other SSRIs,¹⁰ we investigated its effect on Sig-1R expression and on the cellular ER stress responses.     

The Lifetime of UDP-galactose:Ceramide Galactosyltransferase Is Controlled by a Distinct Endoplasmic Reticulum-associated Degradation (ERAD) Regulated by Sigma-1 Receptor Chaperones

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.     

Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca²⁺ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.     

Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival

The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.     

Sigma receptor knockdown augments dysfunction and apoptosis of beta cells induced by palmitate

Sigma-1 receptor (Sig-1R) is located in the endoplasmic reticulum (ER) and clustered on the mitochondria related endoplasmic membranes, which are involved in the regulation of nervous system disease. Here, we designed Sig-1R silence MIN6 cells and studied the influence of Sig-1R silence on beta cells. We showed Sig-1R inactivation in MIN6 cells could not only decrease cell proliferation but also inhibit cell cycle, and this inhibitory effect on cell cycle might be achieved by regulating the FoxM1/Plk1/Cenpa pathway. Moreover, Sig-1R deficiency increased MIN6 cells sensitivity to lipotoxicity, exaggerated palmitate (PA)-induced apoptosis, and impaired insulin secretion. On the other hand, ER chaperone GRP78 and ER proapoptotic molecules CHOP increased in Sig-1R knockdown MIN6 cells. The ATP level decreased and reactive oxygen species (ROS) increased in this kind of cells. Furthermore not only GRP78 and CHOP levels, but also ATP and ROS levels changed more in Sig-1R silence cells after cultured with PA. Therefore, Sig-1R deficiency exaggerated PA induced beta cells apoptosis by aggravating ER stress and mitochondrial dysfunction. Together, our study showed that Sig-1R might influence the proliferation, apoptosis, and function of beta cells.     

Role of sigma-1 receptor C-terminal segment in inositol 1,4,5-trisphosphate receptor activation: constitutive enhancement of calcium signaling in MCF-7 tumor cells

Sigma-1 receptor (sigma-1R) agonists enhance inositol 1,4,5-trisphosphate (IP3)-dependent calcium release from endoplasmic reticulum by inducing dissociation of ankyrin B 220 (ANK 220) from the IP3 receptor (IP3R-3), releasing it from inhibition. MCF-7 breast tumor cells express little or no sigma-1R and were used here to investigate the effect of receptor overexpression and the role of its N- and C-terminal segments in function. We stably expressed intact sigma-1R (amino acids (aa) 1-223; lines 11 and 41), N-fragment (aa 1-100; line K3), or C-fragment (aa 102-223; line sg101). C-fragment expressed as a peripheral membrane-bound protein that was removable from the endoplasmic reticulum membrane by chaotropic salt wash, consistent with lack of a putative transmembrane domain. The expressed sigma-1R, N-fragment, and C-fragment exhibited normal, low affinity, and no [3H](+)-pentazocine binding activity, respectively. All transfected lines showed constitutive enhancement of bradykinin (BDK)-induced calcium release, because of a decrease in BDK ED50 values. Interestingly, sigma-1R and C-fragment had high activities, whereas the N-fragment was much less active. The antagonist BD1063 behaved as an inverse agonist in sigma-1R cells, whereas C-fragment was insensitive to ligand regulation. Like BDK, vasopressin- and ATP-induced calcium release was enhanced with the same pattern in cell lines. Anti-IP3R-3 immunoprecipitates from cells expressing sigma-1R or C-fragment contained significantly less ANK 220 compared with untransfected or N-fragment cells, indicating a higher amount of ankyrin-free IP3R-3. Anti-ankyrin B immunoprecipitates contained sigma-1R or C-fragment, with markedly lower levels of N-fragment present. These results suggest that sigma-1R overexpression drives sigma agonist-independent dissociation of ANK 220 from IP3R-3, resulting in activation. The C-terminal segment plays a key role in the interaction.     

Chronic antidepressants potentiate via sigma-1 receptors the brain-derived neurotrophic factor-induced signaling for glutamate release

Up-regulation of BDNF (brain-derived neurotrophic factor) has been suggested to contribute to the action of antidepressants. However, it is unclear whether chronic treatment with antidepressants may influence acute BDNF signaling in central nervous system neurons. Because BDNF has been shown by us to reinforce excitatory glutamatergic transmission in cultured cortical neurons via the phospholipase-gamma (PLC-gamma)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway (Numakawa, T., Yamagishi, S., Adachi, N., Matsumoto, T., Yokomaku, D., Yamada, M., and Hatanaka, H. (2002) J. Biol. Chem. 277, 6520-6529), we examined in this study the possible effects of pretreatment with antidepressants on the BDNF signaling through the PLC-gamma)/IP3/Ca2+ pathway. Furthermore, because the PLC-gamma/IP3/Ca2+ pathway is regulated by sigma-1 receptors (Hayashi, T., and Su, T. P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 491-496), we examined whether the BDNF signaling is modulated by sigma-1 receptors (Sig-1R). We found that the BDNF-stimulated PLC-gamma activation and the ensued increase in intracellular Ca2+ ([Ca2+]i) were potentiated by pretreatment with imipramine or fluvoxamine, so was the BDNF-induced glutamate release. Furthermore, enhancement of the interaction between PLC-gamma and TrkB (receptor for BDNF) after imipramine pretreatment was observed. Interestingly, BD1047, a potent Sig-1R antagonist, blocked the imipramine-dependent potentiation on the BDNF-induced PLC-gamma activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-gamma activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-gamma/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release.     

Fluvoxamine alleviates paclitaxel-induced neurotoxicity

Paclitaxel (Px) is an effective chemotherapeutic agent for the treatment of various cancers. However, it is often associated with neurological side effects, including chemotherapy-associated cognitive impairment (CACI), such as “chemobrain”. Previously, we reported that endoplasmic reticulum (ER) stress is involved in Px-induced neurotoxicity, and immunoglobulin heavy chain binding protein (BiP) inducer X (BIX) alleviates Px-induced neurotoxicity. However, BIX has not been used in clinical practice yet. We recently reported that fluvoxamine (Flv) alleviates ER stress via induction of sigma-1 receptor (Sig-1R). The purpose of this study was to investigate whether Flv could alleviate Px-induced neurotoxicity in vitro. SK-N-SH cells were pre-treated for 12 h with or without 10 μg/ml Flv followed by treatment with 1 μM Px with or without co-existence of 10 μg/ml Flv for 24 h. To investigate the involvement of Sig-1R in alleviation effect on Px-induced neurotoxicity,1 μM NE100, an antagonist of Sig-1R, was added for 24 h. Neurotoxicity was assessed using the MTS viability assay and ER stress-mediated neurotoxicity was assessed by evaluating the expression of C/EBP homologous protein (CHOP), cleaved caspase 4, and cleaved caspase 3.Pre-treatment with Flv significantly alleviated the induction of CHOP, cleaved caspase 4, and cleaved caspase 3 in SK-N-SH cells. At the same time, pre-treatment with Flv significantly induced Sig-1R in SK-N-SH cells. In addition, viability was significantly higher in Flv-treated cells than in untreated cells, which was reversed by treatment with NE100.Our results suggest that Flv alleviates Px-induced neurotoxicity in part through the induction of Sig-1R. Our findings should contribute to one of the novel approaches for the alleviation of Px-induced neurotoxicity, including chemobrain.     

Antigen retrieval to improve the immunocytochemistry detection of sigma-1 receptors and ER chaperones

Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris–HCl (pH 9.5) containing 6 M urea (80°C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 h effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures.     

Functional assays to define agonists and antagonists of the sigma-2 receptor

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC₅₀ values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC₅₀ values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.     

Knocking Out Sigma-1 Receptors Reveals Diverse Health Problems

Cellular and Molecular Neurobiology - Sigma-1 receptor (Sig-1R) is a protein present in several organs such as brain, lung, and heart. In a cell, Sig-1R is mainly located across the membranes of...     

siRNA-mediated type 1 insulin-like growth factor receptor silencing induces chemosensitization of a human liver cancer cell line with mutant P53

Overexpression of type 1 insulin-like growth factor receptor (IGF1R) contributes to the progression and metastasis of liver cancer, implying that IGF1R gene is a suitable target of RNA interference (RNAi) for liver cancer therapy. To investigate the possible regulation of IGF1R by P53, we examined the level of IGF1R expression in liver cancer cell lines in response to adriamycin. Levels of IGF1R mRNA and protein in cell lines with wild-type P53 decreased dramatically after P53 induction, but no such reduction of IGF1R was observed in cell lines with mutated P53. Inhibition of wild-type P53 in HEPG2 cells by small interfering RNA (siRNA) significantly upregulated the expression of IGF1R. IGF1R inhibition by siRNA in Huh7 cells with mutated P53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of IGF1R, we depressed IGF1R expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 h incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting IGF1R, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase-3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting IGF1R. Our results demonstrate that IGF1R is downregulated by P53, and that siRNA targeting of IGF1R increases liver cancer cells sensitivity to adriamycin and promotes apoptosis. siRNA targeting of IGF1R could be potentially useful for increasing sensitivity to anti-cancer drugs, especially in drug-resistant cells with mutated P53.     

α6 Integrin Transactivates Insulin-like Growth Factor Receptor-1 (IGF-1R) to Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation

The canonical mitochondrial death pathway was first discovered for its role in signaling apoptosis. It has since been found to have a requisite function in differentiation initiation in many cell types including the lens through low level activation of the caspase-3 protease. The ability of this pathway to function as a molecular switch in lens differentiation depends on the concurrent induction of survival molecules in the Bcl-2 and IAP families, induced downstream of an IGF-1R/NFκB coordinate survival signal, to regulate caspase-3 activity. Here we investigated whether α6 integrin signals upstream to this IGF-1R-mediated survival-linked differentiation signal. Our findings show that IGF-1R is recruited to and activated specifically in α6 integrin receptor signaling complexes in the lens equatorial region, where lens epithelial cells initiate their differentiation program. In studies with both α6 integrin knock-out mice lenses and primary lens cell cultures following α6 integrin siRNA knockdown, we show that IGF-1R activation is dependent on α6 integrin and that this transactivation requires Src kinase activity. In addition, without α6 integrin, activation and expression of NFκB was diminished, and expression of Bcl-2 and IAP family members were down-regulated, resulting in high levels of caspase-3 activation. As a result, a number of hallmarks of lens differentiation failed to be induced; including nuclear translocation of Prox1 in the differentiation initiation zone and apoptosis was promoted. We conclude that α6 integrin is an essential upstream regulator of the IGF-1R survival pathway that regulates the activity level of caspase-3 for it to signal differentiation initiation of lens epithelial cells.     

Insulin-like growth factor receptor-1 and nuclear factor κB are crucial survival signals that regulate caspase-3-mediated lens epithelial cell differentiation initiation

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation induction is dependent on the insulin-like growth factor-1 receptor (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling the expression of molecules in the Bcl-2 and inhibitor of apoptosis protein (IAP) families. This effect of IGF-1R was mediated through NFκB, demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFκB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFκB signaling pathway redirected cell fate from differentiation toward apoptosis. These results provided the first evidence that the IGF-1R/NFκB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

Sigma-1受体在慢性疼痛中的调节作用及其神经机制

Sigma-1受体(sigma-1 receptor,Sig-1R)属于配基依赖性的分子伴侣蛋白质,广泛表达于神经系统的多个区域,并可通过结合多种类型的阳离子通道及G蛋白偶联受体(G-protein-coupled receptors,GPCRs)对它们介导的细胞内效应进行调控,或是在内质网和线粒体相关膜结构上对细胞内...     

Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells

Background

Sigma-1 receptors are involved in regulation of neuronal activities presumably through regulation of the activity of ion channels. Sigma-1 receptors also play a role in growth and metastasis of cancerous cells. Intracellular distribution of sigma-1 receptors have been linked to sphingolipid-enriched domains.

Results

We report that in CHO-K1 cells sigma-1 receptors target to focal adhesion contacts (FAC) where they colocalize with Talin and Kv1.4 potassium channels. The levels of sigma-1 receptors in the FAC were significantly increased by application of sigma-1 receptor ligands and by filamentous actin (F-actin) polymerization with phalloidin. The total length of FAC (measured by the focal adhesion marker, talin) was concomitantly increased in the presence of sigma-1 receptors upon phalloidin treatment. Only sigma-1 receptor ligands, however, resulted in an increase of sigma-1 receptors in the FAC, independent of talin. Additionally, a novel approach was utilized to allow an assessment of the half life of endogenous sigma-1 receptors in CHO-K1 cells, which was measured to be at least 72 hours.

Conclusion

Ligand activated sigma-1 receptors translocate into FAC from a pool of receptors stored in ER lipid rafts presumably for inhibition of Kv1.4 channels. Stabilization of actin filaments is likely to be important for targeting sigma-1 receptors to Focal Adhesion Contacts in CHO-K1 cells.