Disturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins-PERK (PEK/EIF2AK3), IRE1, ...and ATF6-is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.
Environmental stresses that disrupt protein homeostasis induce phosphorylation of eIF2, triggering repression of global protein synthesis coincident with preferential translation of ATF4, a ...transcriptional activator of the integrated stress response (ISR). Depending on the extent of protein disruption, ATF4 may not be able to restore proteostatic control and instead switches to a terminal outcome that features elevated expression of the transcription factor CHOP (GADD153/DDIT3). The focus of this study is to define the mechanisms by which CHOP directs gene regulatory networks that determine cell fate. We find that in response to proteasome inhibition, CHOP enhances the expression of a collection of genes encoding transcription regulators, including ATF5, which is preferentially translated during eIF2 phosphorylation. Transcriptional expression of ATF5 is directly induced by both CHOP and ATF4. Knockdown of ATF5 increases cell survival in response to proteasome inhibition, supporting the idea that both ATF5 and CHOP have proapoptotic functions. Transcriptome analysis of ATF5-dependent genes reveals targets involved in apoptosis, including NOXA, which is important for inducing cell death during proteasome inhibition. This study suggests that the ISR features a feedforward loop of stress-induced transcriptional regulators, each subject to transcriptional and translational control, which can switch cell fate toward apoptosis.
Type 1 diabetes (T1D) is a chronic multi-factorial disorder characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. Variations at a large number of genes ...influence susceptibility to spontaneous autoimmune T1D in non-obese diabetic (NOD) mice, one of the most frequently studied animal models for human disease. The genetic analysis of these mice allowed the identification of many insulin-dependent diabetes (Idd) loci and candidate genes, one of them being Cd101. CD101 is a heavily glycosylated transmembrane molecule which exhibits negative-costimulatory functions and promotes regulatory T (Treg) function. It is abundantly expressed on subsets of lymphoid and myeloid cells, particularly within the gastrointestinal tract. We have recently reported that the genotype-dependent expression of CD101 correlates with a decreased susceptibility to T1D in NOD.B6 Idd10 congenic mice compared to parental NOD controls. Here we show that the knockout of CD101 within the introgressed B6-derived Idd10 region increased T1D frequency to that of the NOD strain. This loss of protection from T1D was paralleled by decreased Gr1-expressing myeloid cells and FoxP3+ Tregs and an enhanced accumulation of CD4-positive over CD8-positive T lymphocytes in pancreatic tissues. As compared to CD101+/+ NOD.B6 Idd10 donors, adoptive T cell transfers from CD101-/- NOD.B6 Idd10 mice increased T1D frequency in lymphopenic NOD scid and NOD.B6 Idd10 scid recipients. Increased T1D frequency correlated with a more rapid expansion of the transferred CD101-/- T cells and a lower proportion of recipient Gr1-expressing myeloid cells in the pancreatic lymph nodes. Fewer of the Gr1+ cells in the recipients receiving CD101-/- T cells expressed CD101 and the cells had lower levels of IL-10 and TGF-β mRNA. Thus, our results connect the Cd101 haplotype-dependent protection from T1D to an anti-diabetogenic function of CD101-expressing Tregs and Gr1-positive myeloid cells and confirm the identity of Cd101 as Idd10.
Disruption of protein folding in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a transcriptional and translational control network designed to restore protein ...homeostasis. Central to the UPR is PKR-like ER kinase (PERK/EIF2AK3) phosphorylation of the α subunit of eIF2 (eIF2α∼P), which represses global translation coincident with preferential translation of mRNAs, such as activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP), that serve to implement UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary over a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α∼P, whereas a notable cohort of key regulators are subject to preferential translation. From the latter group, we identified the α isoform of inhibitor of Bruton's tyrosine kinase (IBTKα) as being subject to both translational and transcriptional induction during eIF2α∼P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves stress-induced relief of two inhibitory upstream open reading frames in the 5'-leader of the transcript. Depletion of IBTKα by short hairpin RNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKα is a key regulator in determining cell fate during the UPR.
The antileukemic agent asparaginase triggers the amino acid response (AAR) in the liver by activating the eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2). To ...explore the mechanism by which AAR induction is necessary to mitigate hepatic lipid accumulation and prevent liver dysfunction during continued asparaginase treatment, wild-type and Gcn2 null mice were injected once daily with asparaginase or phosphate buffered saline for up to 14 days. Asparaginase induced mRNA expression of multiple AAR genes and greatly increased circulating concentrations of the metabolic hormone fibroblast growth factor 21 (FGF21) independent of food intake. Loss of Gcn2 precluded mRNA expression and circulating levels of FGF21 and blocked mRNA expression of multiple genes regulating lipid synthesis and metabolism including Fas, Ppara, Pparg, Acadm, and Scd1 in both liver and white adipose tissue. Furthermore, rates of triglyceride export and protein expression of apolipoproteinB-100 were significantly reduced in the livers of Gcn2 null mice treated with asparaginase, providing a mechanistic basis for the increase in hepatic lipid content. Loss of AAR-regulated antioxidant defenses in Gcn2 null livers was signified by reduced Gpx1 gene expression alongside increased lipid peroxidation. Substantial reductions in antithrombin III hepatic expression and activity in the blood of asparaginase-treated Gcn2 null mice indicated liver dysfunction. These results suggest that the ability of the liver to adapt to prolonged asparaginase treatment is influenced by GCN2-directed regulation of FGF21 and oxidative defenses, which, when lost, corresponds with maladaptive effects on lipid metabolism and hemostasis.
Complement, NKT, and NK cells play critical roles in the first line defense against pathogens. Functional roles for both C5a receptors, that is, complement receptor C5a (C5aR) and C5a receptor-like 2 ...(C5L2), in sepsis have been demonstrated. However, the role of C5a in innate lymphocyte activation during sepsis remains elusive. In this article, we show that naive NKT and NK cells already express high levels of C5aR and minor levels of C5L2 mRNA, but no protein. Upon Escherichia coli-induced sepsis, we found C5aR surface expression on subpopulations of NKT and NK cells, suggesting rapid translation into C5aR protein on bacterial encounter. Importantly, significantly increased survival in the absence of C5aR, NKT, and NK cells, but not of C5L2, was associated with reduced IFN-γ and TNF-α serum levels. Sepsis induction in C5aR(+)/C5aR(-) mixed bone marrow chimeras identified cognate engagement of C5aR on NKT cells as an important factor for the recruitment of NKT cells. Furthermore, we found synergistic interaction between C5aR and TLRs enhancing the production of TNF-α and IFN-γ from NKT and NK cells in cocultures with dendritic cells. Our results identify C5aR activation as a novel pathway driving detrimental effects of NKT and NK cells during early experimental sepsis.
Environmental and genetic factors define the susceptibility of an individual to autoimmune disease. Although common genetic pathways affect general immunological tolerance mechanisms in autoimmunity, ...the effects of such genes could vary under distinct immune challenges within different tissues. In this study, we demonstrate this by observing that autoimmune type 1 diabetes-protective haplotypes at the insulin-dependent diabetes susceptibility region 10 (Idd10) introgressed from chromosome 3 of C57BL/6 (B6) and A/J mice onto the NOD background increase the severity of autoimmune primary biliary cirrhosis induced by infection with Novosphingobium aromaticivorans, a ubiquitous alphaproteobacterium, when compared with mice having the NOD and NOD.CAST Idd10 type 1 diabetes-susceptible haplotypes. Substantially increased liver pathology in mice having the B6 and A/J Idd10 haplotypes correlates with reduced expression of CD101 on dendritic cells, macrophages, and granulocytes following infection, delayed clearance of N. aromaticivorans, and the promotion of overzealous IFN-γ- and IL-17-dominated T cell responses essential for the adoptive transfer of liver lesions. CD101-knockout mice generated on the B6 background also exhibit substantially more severe N. aromaticivorans-induced liver disease correlating with increased IFN-γ and IL-17 responses compared with wild-type mice. These data strongly support the hypothesis that allelic variation of the Cd101 gene, located in the Idd10 region, alters the severity of liver autoimmunity induced by N. aromaticivorans.
Abstract only
Phosphorylation of eIF2 (eIF2‐P) lowers protein synthesis to conserve resources and facilitate preferential translation of specific transcripts that promote adaptation to environmental ...stresses. Central to the mechanisms of preferential translation are upstream ORFs (uORFs) embedded within the 5′‐leaders of mRNAs, which serve as “bar codes” that help ribosomes determine the levels of translation of mRNA coding sequences (CDS) during eIF2‐P. We have been studying the functions of uORFs that demarcate translational control and the roles that these preferentially translated genes play in cell adaptation to stress. Preferentially translated GADD34 (PPP1R15A) and constitutively expressed CReP (PPP1R15B) each function to dephosphorylate eIF2‐P and restore protein synthesis. Both GADD34 and CReP mRNAs contain two upstream uORFs, and using biochemical and genetic approaches we show that distinct features of these uORFs are critical for differential CDS translation. In the absence of stress and low eIF2‐P, translation of an inhibitory uORF in GADD34 acts as a barrier by inefficient translation termination that prevents subsequent reinitiation at the GADD34 CDS. Induced eIF2‐P during stress directs ribosome bypass of the inhibitory uORF, leading to enhanced translation of the GADD34 CDS. By comparison, CReP expression occurs independent of eIF2‐P via an uORF that allows for translation reinitiation at the CReP CDS independent of stress conditions. Alterations in the GADD34 uORF not only affect the status of eIF2‐P and translational control, but also cell adaptation to stress. Translation of other preferentially translated genes also feature bypass of uORFs, although by mechanisms featuring different uORF properties. For example, bypass of a single uORF directs preferential translation of CHOP (DDIT3/GADD153), a transcription factor that triggers cell death during periods of extended stress. We discovered that the CHOP uORF serves to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP CDS. Deletion of the CHOP uORF increases the levels and alters the pattern of induced CHOP expression during eIF2‐P. Elevated CHOP expression leads to increased cell death in response to stress. Finally, we determined that bypass of uORFs in IBTKalpha enhances translation of the CDS in response to eIF2‐P. Elevated levels of IBTKalpha localize to endoplasmic reticulum exit sites and facilitate initiation of autophagy. These examples and others indicate that the proper mixing and matching of uORFs with distinct properties are central for directing gene‐specific translation during eIF2‐P. Preferentially translated genes are critical for the timing and extent of translation and transcription, and alter metabolism and reclamation processes such as autophagy, which together can determine cell survival during environmental stresses.
Support or Funding Information
NIH GM049164
We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes ...susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.
Type 1 diabetes (T1D) is a chronic multi-factorial disorder characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. Variations at a large number of genes ...influence susceptibility to spontaneous autoimmune T1D in non-obese diabetic (NOD) mice, one of the most frequently studied animal models for human disease. The genetic analysis of these mice allowed the identification of many insulin-dependent diabetes (Idd) loci and candidate genes, one of them being Cd101. CD101 is a heavily glycosylated transmembrane molecule which exhibits negative-costimulatory functions and promotes regulatory T (Treg) function. It is abundantly expressed on subsets of lymphoid and myeloid cells, particularly within the gastrointestinal tract. We have recently reported that the genotype-dependent expression of CD101 correlates with a decreased susceptibility to T1D in NOD.B6 Idd10 congenic mice compared to parental NOD controls. Here we show that the knockout of CD101 within the introgressed B6-derived Idd10 region increased T1D frequency to that of the NOD strain. This loss of protection from T1D was paralleled by decreased Gr1-expressing myeloid cells and FoxP3.sup.+ Tregs and an enhanced accumulation of CD4-positive over CD8-positive T lymphocytes in pancreatic tissues. As compared to CD101.sup.+/+ NOD.B6 Idd10 donors, adoptive T cell transfers from CD101.sup.-/- NOD.B6 Idd10 mice increased T1D frequency in lymphopenic NOD scid and NOD.B6 Idd10 scid recipients. Increased T1D frequency correlated with a more rapid expansion of the transferred CD101.sup.-/- T cells and a lower proportion of recipient Gr1-expressing myeloid cells in the pancreatic lymph nodes. Fewer of the Gr1.sup.+ cells in the recipients receiving CD101.sup.-/- T cells expressed CD101 and the cells had lower levels of IL-10 and TGF-beta mRNA. Thus, our results connect the Cd101 haplotype-dependent protection from T1D to an anti-diabetogenic function of CD101-expressing Tregs and Gr1-positive myeloid cells and confirm the identity of Cd101 as Idd10.
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