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1.  Endoplasmic Reticulum Stress in the β-Cell Pathogenesis of Type 2 Diabetes 
Experimental Diabetes Research  2011;2012:618396.
Type 2 diabetes is a complex metabolic disorder characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency by β-cell failure. Even if the mechanisms underlying the pathogenesis of β-cell failure are still under investigation, recent increasing genetic, experimental, and clinical evidence indicate that hyperactivation of the unfolded protein response (UPR) to counteract metabolic stresses is closely related to β-cell dysfunction and apoptosis. Signaling pathways of the UPR are “a double-edged sword” that can promote adaptation or apoptosis depending on the nature of the ER stress condition. In this paper, we summarized our current understanding of the mechanisms and components related to ER stress in the β-cell pathogenesis of type 2 diabetes.
doi:10.1155/2012/618396
PMCID: PMC3170700  PMID: 21915177
2.  Efficient lytic induction of kaposi's sarcoma-associated herpesvirus (KSHV) by the anthracyclines 
Oncotarget  2014;5(18):8515-8527.
Lytic induction of latent Kaposi's sarcoma-associated herpesvirus (KSHV) has been considered as a therapeutic option for efficient treatment of several KSHV-associated malignancies. Here, we developed a robust high-throughput screening system that allows an easy and quantitative measurement of lytic induction of latent KSHV and discovered three anthracyclines as potent inducers from screen of FDA-approved drugs. Lytic induction of latent KSHV by three compounds was verified by the significant induction of lytic genes and subsequent production of infectious KSHV. Importantly, lytic induction by three compounds was much more efficient than that by sodium butyrate, a well-characterized inducer of KSHV lytic cycle. Mechanistically, the anthracyclines caused lytic induction of KSHV through apoptosis induced by their DNA intercalation rather than topoisomerase II inhibition. Consequently, our results clearly demonstrated a role of anthracyclines as effective lytic inducers of KSHV and also provided a molecular basis of their use for efficient treatment of diseases associated with KSHV infection.
PMCID: PMC4226701  PMID: 25237786
Kaposi's sarcoma-associated herpesvirus (KSHV); Lytic induction; Anthracyclines; Apoptosis; DNA intercalation
3.  Orphan Nuclear Receptor Errγ Induces C-Reactive Protein Gene Expression through Induction of ER-Bound Bzip Transmembrane Transcription Factor CREBH 
PLoS ONE  2014;9(1):e86342.
The orphan nuclear receptor estrogen-related receptor-γ (ERRγ) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE) in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions.
doi:10.1371/journal.pone.0086342
PMCID: PMC3899246  PMID: 24466039
4.  ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death 
Nature cell biology  2013;15(5):481-490.
Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.
doi:10.1038/ncb2738
PMCID: PMC3692270  PMID: 23624402
5.  Endoplasmic Reticulum Stress and Type 2 Diabetes 
Annual review of biochemistry  2012;81:767-793.
Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death.
doi:10.1146/annurev-biochem-072909-095555
PMCID: PMC3684428  PMID: 22443930
unfolded protein response; ER stress; free fatty acid; glucose; pancreatic β-cell
6.  Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response 
Nucleic Acids Research  2013;41(14):6960-6974.
Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane–bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane–bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.
doi:10.1093/nar/gkt429
PMCID: PMC3737538  PMID: 23716639
7.  The unfolded protein response is required to maintain the integrity of the ER, prevent oxidative stress, and preserve differentiation in β-cells 
Diabetes, obesity & metabolism  2010;12(Suppl 2):99-107.
Diabetes is an epidemic of worldwide proportions caused by β-cell failure. Nutrient fluctuations and insulin resistance drive β–cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signaling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). PERK signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the alpha subunit (eIF2α) to attenuate protein synthesis, thereby reducing the biosynthetic burden. B–cells uniquely require PERK-mediated phosphorylation of eIF2α to preserve cell function. Unabated protein synthesis in β–cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of β–cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT-enhancer binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases β–cell mass and prevents β–cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER cause oxidative stress and should encourage the development of novel strategies to treat diabetes.
doi:10.1111/j.1463-1326.2010.01281.x
PMCID: PMC3127455  PMID: 21029306
eukaryotic initiation factor 2; PERK; CHOP; antioxidant; apoptosis; translation; mitochondria; protein folding
8.  UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators 
Developmental cell  2008;15(6):829-840.
Summary
The unfolded protein response (UPR) is linked to metabolic dysfunction, yet it is not known how ER disruption might influence metabolic pathways. Using a multilayered genetic approach, we find that mice with genetic ablations of either ER stress sensing pathways (ATF6α, eIF2α, IRE1α), or of ER quality control (p58IPK), share a common dysregulated response to ER stress that includes the development of microvesicular steatosis. The rescue of ER protein processing capacity by the combined action of UPR pathways during stress prevents the suppression of a subset of metabolic transcription factors that regulate lipid homeostasis. This suppression occurs in part by unresolved ER stress perpetuating expression of the transcriptional repressor CHOP. As a consequence, metabolic gene expression networks are directly responsive to ER homeostasis. These results reveal an unanticipated direct link between ER homeostasis and the transcriptional regulation of metabolism and suggest mechanisms by which ER stress might underlie microvesicular steatosis.
doi:10.1016/j.devcel.2008.10.015
PMCID: PMC2923556  PMID: 19081072
9.  ATF6α induces XBP1-independent expansion of the endoplasmic reticulum 
Journal of cell science  2009;122(Pt 10):1626-1636.
Summary
A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6α (ATF6α), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6α drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6α induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6α and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.
doi:10.1242/jcs.045625
PMCID: PMC2680102  PMID: 19420237
Endoplasmic reticulum biogenesis; Unfolded protein response; ATF6α; XBP1; Lipid biosynthesis
10.  Translation attenuation through eIF2α phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells 
Cell metabolism  2009;10(1):13-26.
SUMMARY
Accumulation of unfolded protein within the endoplasmic reticulum (ER) lumen attenuates mRNA translation through activation of the protein kinase PERK and subsequent phosphorylation of eukaryotic initiation factor 2 on Ser51 of the alpha subunit (eIF2α). Genetic disruption of the PERK/eIF2α pathway in humans and mice produces severe pancreatic beta cell deficiency and post-natal lethality. To elucidate the role of eIF2α phosphorylation in beta cells, we have rescued the lethality of homozygous eIF2α Ser51Ala mice by expression of a loxP-flanked wild-type eIF2α transgene. Beta cell-specific transgene deletion to prevent eIF2α phosphorylation caused a severe diabetic phenotype due to heightened, unregulated proinsulin translation, defective intracellular trafficking of secretory and plasma membrane proteins, increased oxidative damage, reduced expression of stress response and beta cell-specific genes, and apoptosis. However, glucose intolerance and beta cell death in these mice were attenuated by antioxidant treatment. We conclude that phosphorylation of eIF2α coordinately attenuates mRNA translation, prevents oxidative stress, and optimizes ER protein folding to support insulin production in the beta cell. These findings that show increased proinsulin synthesis causes oxidative stress leading to beta cell failure may reflect events in the beta cell loss associated with insulin resistance in type 2 diabetes.
doi:10.1016/j.cmet.2009.06.002
PMCID: PMC2742645  PMID: 19583950
11.  Sequestration of TRAF2 into Stress Granules Interrupts Tumor Necrosis Factor Signaling under Stress Conditions 
Molecular and Cellular Biology  2005;25(6):2450-2462.
The cellular stress response (SR) is a phylogenetically conserved protection mechanism that involves inhibition of protein synthesis through recruitment of translation factors such as eIF4G into insoluble stress granules (SGs) and blockade of proinflammatory responses by interruption of the signaling pathway from tumor necrosis factor alpha (TNF-α) to nuclear factor-κB (NF-κB) activation. However, the link between these two physiological phenomena has not been clearly elucidated. Here we report that eIF4GI, which is a scaffold protein interacting with many translation factors, interacts with TRAF2, a signaling molecule that plays a key role in activation of NF-κB through TNF-α. These two proteins colocalize in SGs during cellular exposure to stress conditions. Moreover, TRAF2 is absent from TNFR1 complexes under stress conditions even after TNF-α treatment. This suggests that stressed cells lower their biological activities by sequestration of translation factors and TRAF2 into SGs through a protein-protein interaction.
doi:10.1128/MCB.25.6.2450-2462.2005
PMCID: PMC1061607  PMID: 15743837
12.  Polypyrimidine Tract-Binding Protein Enhances the Internal Ribosomal Entry Site-Dependent Translation of p27Kip1 mRNA and Modulates Transition from G1 to S Phase 
Molecular and Cellular Biology  2005;25(4):1283-1297.
The p27Kip1 protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27Kip1 is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of p27Kip1 mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27Kip1 mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27Kip1 mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27Kip1 mRNA. Moreover, the G1 phase in the cell cycle (which is maintained in part by p27Kip1) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27Kip1 protein synthesis during differentiation. The IRES activity of p27Kip1 mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27Kip1 mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27Kip1 mRNA.
doi:10.1128/MCB.25.4.1283-1297.2005
PMCID: PMC548013  PMID: 15684381
13.  Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro 
Journal of Virology  2002;76(5):2529-2542.
The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA.
PMCID: PMC135932  PMID: 11836431
14.  La autoantigen enhances translation of BiP mRNA 
Nucleic Acids Research  2001;29(24):5009-5016.
Translational initiation of the human BiP mRNA is directed by an internal ribosomal entry site (IRES) located in the 5′-untranslated region (5′-UTR). In order to understand the mechanism of the IRES-dependent translation of BiP mRNA, cellular proteins interacting with the BiP IRES were investigated. La autoantigen, which augments the translation of polioviral mRNA and hepatitis C viral mRNA, bound specifically to the second half of the 5′-UTR of the BiP IRES and enhanced translation of BiP mRNA in both in vitro and in vivo assays. This finding suggests that cellular and viral IRESs containing very different RNA sequences may share a common mechanism of translation.
PMCID: PMC97601  PMID: 11812831

Results 1-14 (14)