Barbara C. McGrath

The Combined Effects of Tryptophan Starvation and Tryptophan Catabolites Down-Regulate T Cell Receptor ζ-Chain and Induce a Regulatory Phenotype in Naive T Cells

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that the short-term, combined effects of tryptophan deprivation and tryptophan catabolites result in GCN2 kinase-dependent down-regulation of the TCR ζ-chain in murine CD8+ T cells. TCR ζ down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25− T cells via TGF-β induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69−, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells.

Reelin Binds α3β1 Integrin and Inhibits Neuronal Migration

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.

The PERK Eukaryotic Initiation Factor 2α Kinase Is Required for the Development of the Skeletal System, Postnatal Growth, and the Function and Viability of the Pancreas

ABSTRACT Phosphorylation of eukaryotic initiation factor 2α (eIF-2α) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2α (eIF-2α[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2α. Among the four eIF-2α kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2α[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk−/− mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk−/− mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.

Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response

ABSTRACT In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.

Phosphorylation of the α Subunit of Eukaryotic Initiation Factor 2 Is Required for Activation of NF-κB in Response to Diverse Cellular Stresses

ABSTRACT Nuclear factor κB (NF-κB) serves to coordinate the transcription of genes in response to diverse environmental stresses. In this report we show that phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2) is fundamental to the process by which many stress signals activate NF-κB. Phosphorylation of this translation factor is carried out by a family of protein kinases that each respond to distinct stress conditions. During impaired protein folding and assembly in the endoplasmic reticulum (ER), phosphorylation of eIF2α by PEK (Perk or EIF2AK3) is essential for induction of NF-κB transcriptional activity. The mechanism by which NF-κB is activated during ER stress entails the release, but not the degradation, of the inhibitory protein IκB. During amino acid deprivation, phosphorylation of eIF2α by GCN2 (EIF2AK4) signals the activation of NF-κB. Furthermore, inhibition of general translation or transcription by cycloheximide and actinomycin D, respectively, elicits the eIF2α phosphorylation required for induction of NF-κB. Together, these studies suggest that eIF2α kinases monitor and are activated by a range of stress conditions that affect transcription and protein synthesis and assembly, and the resulting eIFα phosphorylation is central to activation of the NF-κB. The absence of NF-κB-mediated transcription and its antiapoptotic function provides an explanation for why eIF2α kinase deficiency in diseases such as Wolcott-Rallison syndrome leads to cellular apoptosis and disease.

The GCN2 eIF2α Kinase Is Required for Adaptation to Amino Acid Deprivation in Mice

ABSTRACT The GCN2 eIF2α kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2s function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2 −/− knockout strain of mice. Gcn2 −/− mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2 −/− mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2 −/− mice failed to show the normal induction of eIF2α phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2α and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2 −/− mice.

PERK (eIF2α kinase) is required to activate the stress-activated MAPKs and induce the expression of immediate-early genes upon disruption of ER calcium homoeostasis

The eIF2alpha (eukaryotic initiation factor-2alpha) kinase PERK (doublestranded RNA-activated protein kinase-like ER kinase) is essential for the normal function of highly secretory cells in the pancreas and skeletal system, as well as the UPR (unfolded protein response) in mammalian cells. To delineate the regulatory machinery underlying PERK-dependent stress-responses, gene profiling was employed to assess global changes in gene expression in PERK-deficient MEFs (mouse embryonic fibroblasts). Several IE (immediate-early) genes, including c-myc, c-jun, egr-1 (early growth response factor-1), and fra-1 (fos-related antigen-1), displayed PERK-dependent expression in MEFs upon disruption of calcium homoeostasis by inhibiting the ER (endoplasmic reticulum) transmembrane SERCA (sarcoplasmic/ER Ca2+-ATPase) calcium pump. Induction of c-myc and egr-1 by other reagents that elicit the UPR, however, showed variable dependence upon PERK. Induction of c-myc expression by thapsigargin was shown to be linked to key signalling enzymes including PLC (phospholipase C), PI3K (phosphatidylinositol 3-kinase) and p38 MAPK (mitogen-activated protein kinase). Analysis of the phosphorylated status of major components in MAPK signalling pathways indicated that thapsigargin and DTT (dithiothreitol) but not tunicamycin could trigger the PERK-dependent activation of JNK (c-Jun N-terminal kinase) and p38 MAPK. However, activation of JNK and p38 MAPK by non-ER stress stimuli including UV irradiation, anisomycin, and TNF-alpha (tumour necrosis factor-alpha) was found to be independent of PERK. PERK plays a particularly important role in mediating the global cellular response to ER stress that is elicited by the depletion of calcium from the ER. We suggest that this specificity of PERK function in the UPR is an extension of the normal physiological function of PERK to act as a calcium sensor in the ER.

PERK (EIF2AK3) Regulates Proinsulin Trafficking and Quality Control in the Secretory Pathway

OBJECTIVE Loss-of-function mutations in Perk (EIF2AK3) result in permanent neonatal diabetes in humans (Wolcott-Rallison Syndrome) and mice. Previously, we found that diabetes associated with Perk deficiency resulted from insufficient proliferation of β-cells and from defects in insulin secretion. A substantial fraction of PERK-deficient β-cells display a highly abnormal cellular phenotype characterized by grossly distended endoplasmic reticulum (ER) and retention of proinsulin. We investigated over synthesis, lack of ER-associated degradation (ERAD), and defects in ER to Golgi trafficking as possible causes. RESEARCH DESIGN AND METHODS ER functions of PERK were investigated in cell culture and mice in which Perk was impaired or gene dosage modulated. The Ins2+/Akita mutant mice were used as a model system to test the role of PERK in ERAD. RESULTS We report that loss of Perk function does not lead to uncontrolled protein synthesis but impaired ER-to-Golgi anterograde trafficking, retrotranslocation from the ER to the cytoplasm, and proteasomal degradation. PERK was also shown to be required to maintain the integrity of the ER and Golgi and processing of ATF6. Moreover, decreasing Perk dosage surprisingly ameliorates the progression of the Akita mutants toward diabetes. CONCLUSIONS PERK is a positive regulator of ERAD and proteasomal activity. Reducing PERK activity ameliorates the progression of diabetes in the Akita mouse, whereas increasing PERK dosage hastens its progression. We speculate that PERK acts as a metabolic sensor in the insulin-secreting β-cells to modulate the trafficking and quality control of proinsulin in the ER relative to the physiological demands for circulating insulin.

PERK is essential for neonatal skeletal development to regulate osteoblast proliferation and differentiation

Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonatal developmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, at the cellular and molecular level in PERK‐deficient mice demonstrated that neonatal Perk−/− mice are severely osteopenic, which is caused by a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaired differentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators of osteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A, Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type I collagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagen production and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development and osteoblast biology. J. Cell. Physiol. 217: 693–707, 2008.

PERK eIF2 alpha kinase is required to regulate the viability of the exocrine pancreas in mice

BackgroundDeficiency of the PERK eIF2α kinase in humans and mice results in postnatal exocrine pancreatic atrophy as well as severe growth and metabolic anomalies in other organs and tissues. To determine if the exocrine pancreatic atrophy is due to a cell-autonomous defect, the Perk gene was specifically ablated in acinar cells of the exocrine pancreas in mice.ResultsWe show that expression of PERK in the acinar cells is required to maintain their viability but is not required for normal protein synthesis and secretion. Exocrine pancreatic atrophy in PERK-deficient mice was previously attributed to uncontrolled ER-stress followed by apoptotic cell death based on studies in cultured fibroblasts. However, we have found no evidence for perturbations in the endoplasmic reticulum or ER-stress and show that acinar cells succumb to a non-apoptotic form of cell death, oncosis, which is associated with a pronounced inflammatory response and induction of the pancreatitis stress response genes. We also show that mice carrying a knockout mutation of PERKs downstream target, ATF4, exhibit pancreatic deficiency caused by developmental defects and that mice ablated for ATF4s transcriptional target CHOP have a normal exocrine pancreas.ConclusionWe conclude that PERK modulates secretory capacity of the exocrine pancreas by regulating cell viability of acinar cells.