scholarly journals Human TSC-associated renal angiomyolipoma cells are hypersensitive to ER stress

2012 ◽  
Vol 303 (6) ◽  
pp. F831-F844 ◽  
Author(s):  
Brian J. Siroky ◽  
Hong Yin ◽  
Justin T. Babcock ◽  
Lu Lu ◽  
Anna R. Hellmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Caspase 3 ◽  
Renal Involvement ◽  
Mammalian Target Of Rapamycin ◽  
Protein Translation ◽  
Cell Number ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Renal Angiomyolipoma

Tuberous sclerosis complex (TSC), an inherited tumor predisposition syndrome associated with mutations in TSC1 or TSC2, affects ∼1 in 6,000 individuals. Eighty percent of TSC patients develop renal angiomyolipomas, and renal involvement is a major contributor to patient morbidity and mortality. Recent work has shown that mammalian target of rapamycin complex 1 (mTORC1) inhibition caused angiomyolipoma shrinkage but that this treatment may cause cytostatic not a cytotoxic effect. Endoplasmic reticulum (ER) stress can develop in TSC-associated cells due to mTORC1-driven protein translation. We hypothesized that renal angiomyolipoma cells experience ER stress that can be leveraged to result in targeted cytotoxicity. We used immortalized human angiomyolipoma cells stably transfected with empty vector or TSC2 (encoding tuberin). Using cell number quantification and cell death assays, we found that mTORC1 inhibition with RAD001 suppressed angiomyolipoma cell proliferation in a cytostatic manner. Angiomyolipoma cells exhibited enhanced sensitivity to proteasome inhibitor-induced ER stress compared with TSC2-rescued cells. After proteasome inhibition with MG-132, Western blot analyses showed greater induction of C/EBP-homologous protein (CHOP) and more poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage, supporting ER stress-induced apoptosis. Live cell numbers also were decreased and cell death increased by MG-132 in angiomyolipoma cells compared with TSC2 rescued. Intriguingly, while pretreatment of angiomyolipoma cells with RAD001 attenuated CHOP and BiP induction, apoptotic markers cleaved PARP and caspase-3 and eukaryotic translation initiation factor 2α phosphorylation were increased, along with evidence of increased autophagy. These results suggest that human angiomyolipoma cells are uniquely susceptible to agents that exacerbate ER stress and that additional synergy may be achievable with targeted combination therapy.

scholarly journals Eukaryotic translation initiation factor 2A protects pancreatic beta cells during endoplasmic reticulum stress while rescuing translation inhibition

2021 ◽  
Author(s):  
Evgeniy Panzhinskiy ◽  
Søs Skovsø ◽  
Haoning Cen ◽  
Kwan Chu ◽  
Kate MacDonald ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Translation ◽  
Human Islets ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Related Factor ◽  
Min6 Cells ◽  
Primary Mouse ◽  
Mouse Islets

Abstract The endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, cell specific EIF2A viral overexpression reduced ER stress, improved insulin secretion, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved in protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for cell protection and the circumvention of EIF2S1-mediated translational repression.

scholarly journals Mitogen-Inducible Gene 6 Triggers Apoptosis and Exacerbates ER Stress-Induced β-Cell Death

2013 ◽  
Vol 27 (1) ◽  
pp. 162-171 ◽  
Author(s):  
Yi-Chun Chen ◽  
E. Scott Colvin ◽  
Bernhard F. Maier ◽  
Raghavendra G. Mirmira ◽  
Patrick T. Fueger
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Caspase 3 ◽  
Cell Mass ◽  
Growth Factor Receptor ◽  
Protein Translation ◽  
Β Cells ◽  
Β Cell ◽  
Inducible Gene

The increased insulin secretory burden placed on pancreatic β-cells during obesity and insulin resistance can ultimately lead to β-cell dysfunction and death and the development of type 2 diabetes. Mitogen-inducible gene 6 (Mig6) is a cellular stress-responsive protein that can negatively regulate the duration and intensity of epidermal growth factor receptor signaling and has been classically viewed as a molecular brake for proliferation. In this study, we used Mig6 heterozygous knockout mice (Mig6+/−) to study the role of Mig6 in regulating β-cell proliferation and survival. Surprisingly, the proliferation rate of Mig6+/− pancreatic islets was lower than wild-type islets despite having comparable β-cell mass and glucose tolerance. We thus speculated that Mig6 regulates cellular death. Using adenoviral vectors to overexpress or knockdown Mig6, we found that caspase 3 activation during apoptosis was dependent on the level of Mig6. Interestingly, Mig6 expression was induced during endoplasmic reticulum (ER) stress, and its protein levels were maintained throughout ER stress. Using polyribosomal profiling, we identified that Mig6 protein translation was maintained, whereas the global protein translation was inhibited during ER stress. In addition, Mig6 overexpression exacerbated ER stress-induced caspase 3 activation in vitro. In conclusion, Mig6 is transcriptionally up-regulated and resistant to global translational inhibition during stressed conditions in β-cells and mediates apoptosis in the form of caspase 3 activation. The sustained production of Mig6 protein exacerbates ER stress-induced β-cell death. Thus, preventing the induction, translation, and/or function of Mig6 is warranted for increasing β-cell survival.

scholarly journals Phosphorylation of Eukaryotic Translation Initiation Factor 2α Coordinates rRNA Transcription and Translation Inhibition during Endoplasmic Reticulum Stress

2009 ◽  
Vol 29 (15) ◽  
pp. 4295-4307 ◽  
Author(s):  
Jenny B. DuRose ◽  
Donalyn Scheuner ◽  
Randal J. Kaufman ◽  
Lawrence I. Rothblum ◽  
Maho Niwa
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosome Biogenesis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rrna Synthesis ◽  
Cell Physiology ◽  
Rrna Transcription

ABSTRACT The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting ∼90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2α, suggesting that this pathway may be broadly utilized by stresses that activate eIF2α kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.

scholarly journals S6 kinase 1 knockout inhibits uninephrectomy- or diabetes-induced renal hypertrophy

2009 ◽  
Vol 297 (3) ◽  
pp. F585-F593 ◽  
Author(s):  
Jian-Kang Chen ◽  
Jianchun Chen ◽  
George Thomas ◽  
Sara C. Kozma ◽  
Raymond C. Harris
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Cell Number ◽  
Northern Blotting ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type ◽  
Minimal Cell ◽  
Renal Hypertrophy ◽  
S6 Kinase ◽  
Compensatory Renal Hypertrophy

Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiological renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild-type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1−/− mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate upregulation of S6K2 expression in the kidneys of S6K1−/− mice. Phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1, another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1−/− and S6K1+/+ littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1−/− mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60–70% of the hypertrophy seen in wild-type mice but failed to prevent the ∼10% hypertrophy seen in S6K1−/− mice in response to uninephrectomy (UNX) although it did inhibit the basal rpS6 phosphorylation. Thus the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNX- and diabetes-mediated mTOR activation can selectively activate S6K1 without activating S6K2.

scholarly journals Eukaryotic translation initiation factor 2A protects pancreatic beta cells during endoplasmic reticulum stress while rescuing translation inhibition

2021 ◽  
Author(s):  
Evgeniy Panzhinskiy ◽  
Søs Skovsø ◽  
Haoning Howard Cen ◽  
Kwan Yi Chu ◽  
Kate MacDonald ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Translation ◽  
Human Islets ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Β Cell ◽  
Min6 Cells ◽  
Primary Mouse ◽  
Mouse Islets

ABSTRACTThe endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide β cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in β cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1α) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, β cell specific EIF2A viral overexpression reduced ER stress, improved insulin secretion, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved in protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for β cell protection and the circumvention of EIF2S1-mediated translational repression.

Xanthohumol Induces Apoptosis in B-CLL In Vitro by Sustained Endoplasmic Reticulum Stress and Inhibition of NFκ-B.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4979-4979
Author(s):  
Sofie Lust ◽  
Barbara Vanhoecke ◽  
Mireille Van Gele ◽  
Mary Kaileh ◽  
Jerina Boelens ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Caspase 3 ◽  
Protein Translation ◽  
Mrna Levels ◽  
Induced Apoptosis ◽  
Sustained Phosphorylation

Abstract Introduction Correct folding of new proteins is supervised in the endoplasic reticulum (ER) unfolded protein response (UPR). Misfolded proteins recruit the chaperone Grp78 that is thereby released from the transcription factors ATF6, IRE-1 leading to compensatory increase in Grp78, and PERK, leading to phosphorylation of eIF2α and block of further protein translation. UPR overload leads to ER stress and cell death. Targeting the endoplasmic reticulum (ER) is a new strategy explored in B-CLL. The hop-derived chalcone Xanthohumol (X) has been characterized as a ‘broad-spectrum’ cancer chemopreventive agent. Recently, we demonstrated that X induces dose- and time-dependent cell death of MCF7/6 breast cancer cells accompanied by ER stress. X induces apoptosis and cleavage of poly(ADP)-ribose-polymerase (PARP) in B-CLL in vitro. The present study investigates the branches of the UPR in relation to X induced apoptosis of B-CLL cells. Materials and methods. Lymphocytes were isolated by Lymphoprep from 15 patients with B-CLL after informed consent. CD19 positive cells were selected by EasySep positive selection kit. Apoptosis was assessed by flow-cytometry (AnnexinV-PI). Western Blotting was used for Grp78, ATF6, XBP1, phospho-eIF2a, eIF2a, ATF4, CHOP, phospho-IKK, IKK, PARP, caspase-9, -8, -7, -4, cleaved caspase-3, mcl-1, bcl-xL, bax, bak, and bid. NF-kB activity was assessed by EMSA. Quantitative RT-PCR was performed to analyze Grp78 mRNA levels. Bcl-2 protein level was detected by flow cytometry and reactive oxygen species (ROS) by fluorescence microscopy. Results and conclusion X induced an upregulation of Grp78 mRNA levels which was not translated in an increase in protein. X treatment stimulated a rapid and sustained phosphorylation of eIF2a, suggesting the involvement of PERK. In contrast, the ER-stress transducers ATF6 and IRE1 were not activated. X-induced ER stress was associated with strong induction of the pro-apoptotic protein CHOP and inhibition of the NF-kB pathway. Furthermore, the pro-apoptotic effect of X was accompanied by an accumulation of ROS, a downregulation of the anti-apoptotic proteins mcl-1, bcl-xL, bcl-2 and processing of caspase-3, -7 and -9.In conclusion, the chalcone X is capable of inducing cell death with down-regulation of bcl-2, mcl-1, bcl-xL, and activation of the caspase cascade. This is accompanied by ER-stress as evidenced by the upregulation of Grp78 mRNA levels, induction of a rapid and sustained phosphorylation of eIF2a, upregulation of CHOP, and inhibition of the NF-kB signaling.

scholarly journals Inhibition of eEF2K synergizes with glutaminase inhibitors or 4EBP1 depletion to suppress growth of triple-negative breast cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
YoungJun Ju ◽  
Yaacov Ben-David ◽  
Daniela Rotin ◽  
Eldad Zacksenhaus
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Translation Initiation ◽  
Triple Negative ◽  
Elongation Factor ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Related Factors ◽  
Metabolic Conditions

AbstractThe eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the ‘Collagen containing extracellular matrix’ pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.

Update on Phytochemical and Biological Studies on Rocaglate Derivatives from Aglaia Species

Planta Medica ◽  
2021 ◽  
Author(s):  
Garima Agarwal ◽  
Long-Sheng Chang ◽  
Djaja Doel Soejarto ◽  
A. Douglas Kinghorn
Keyword(s):  
Biological Activities ◽  
Chemical Constituents ◽  
Protein Translation ◽  
Biological Properties ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Ability To Act ◽  
Biological Studies ◽  
Phytochemical Constituents

AbstractWith about 120 species, Aglaia is one of the largest genera of the plant family Meliaceae (the mahogany plants). It is native to the tropical rainforests of the Indo-Australian region, ranging from India and Sri Lanka eastward to Polynesia and Micronesia. Various Aglaia species have been investigated since the 1960s for their phytochemical constituents and biological properties, with the cyclopenta[b]benzofurans (rocaglates or flavaglines) being of particular interest. Phytochemists, medicinal chemists, and biologists have conducted extensive research in establishing these secondary metabolites as potential lead compounds with antineoplastic and antiviral effects, among others. The varied biological properties of rocaglates can be attributed to their unusual structures and their ability to act as inhibitors of the eukaryotic translation initiation factor 4A (eIF4A), affecting protein translation. The present review provides an update on the recently reported phytochemical constituents of Aglaia species, focusing on rocaglate derivatives. Furthermore, laboratory work performed on investigating the biological activities of these chemical constituents is also covered.

scholarly journals Palmitate Causes Endoplasmic Reticulum Stress and Apoptosis in Human Mesenchymal Stem Cells: Prevention by AMPK Activator

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5275-5284 ◽  
Author(s):  
Jun Lu ◽  
Qinghua Wang ◽  
Lianghu Huang ◽  
Huiyue Dong ◽  
Lingjing Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endoplasmic Reticulum ◽  
Mesenchymal Stem Cells ◽  
Protein Kinase ◽  
Er Stress ◽  
Human Mesenchymal Stem Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Human Umbilical Cord ◽  
Amp Activated Protein Kinase

Abstract Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

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