General Inhibition Of Protein Synthesis Research Articles

Homozygous knockout of eEF2K alleviates cognitive deficits in APP/PS1 Alzheimer's disease model mice independent of brain amyloid β pathology.

Maintenance of memory and synaptic plasticity depends on de novo protein synthesis, and accumulating evidence implicates a role of dysregulated mRNA translation in cognitive impairments associated with Alzheimer’s disease (AD). Accumulating evidence demonstrates hyper-phosphorylation of translation factor eukaryotic elongation factor 2 (eEF2) in the hippocampi of human AD patients as well as transgenic AD model mice. Phosphorylation of eEF2 (at the Thr 56 site) by its only known kinase, eEF2K, leads to inhibition of general protein synthesis. A recent study suggests that amyloid β (Aβ)-induced neurotoxicity could be associated with an interaction between eEF2 phosphorylation and the transcription factor nuclear erythroid 2-related factor (NRF2)-mediated antioxidant response. In this brief communication, we report that global homozygous knockout of the eEF2K gene alleviates deficits of long-term recognition and spatial learning in a mouse model of AD (APP/PS1). Moreover, eEF2K knockout does not alter brain Aβ pathology in APP/PS1 mice. The hippocampal NRF2 antioxidant response in the APP/PS1 mice, measured by expression levels of nicotinamide adenine dinucleotide plus hydrogen (NADPH) quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), is ameliorated by suppression of eEF2K signaling. Together, the findings may contribute to our understanding of the molecular mechanisms underlying AD pathogenesis, indicating that suppression of eEF2K activity could be a beneficial therapeutic option for this devastating neurodegenerative disease.

Memory impairments in Tg19959 and APP/PS1 AD model mice are alleviated by eEF2K inhibitor A‐484954

AbstractBackgroundMounting evidence indicates synaptic failure as an early and key event in Alzheimer’s Disease (AD) pathophysiology. Maintenance of long‐term memory and synaptic plasticity requires de novo protein synthesis. Phosphorylation of mRNA translation factor eukaryotic elongation factor 2 (eEF2) by its kinase eEF2K results in inhibition of general protein synthesis. Previous studies have shown elevated levels of eEF2 phosphorylation in post mortem AD human brain tissue and in AD mouse models. Here we investigated whether suppression of eEF2 phosphorylation via eEF2K inhibitor A‐484954 can alleviate AD‐associated synaptic failure and memory impairmentsMethodAged Tg19959 mice (6‐9 months), APP/PS1 (12‐16 months) and age‐matched controls were treated with a subcutaneous pellet containing A‐484954 or vehicle. The pellet continuously releases treatment over 30 days. Starting two weeks after pellet placement, the mice underwent cognitive assessment via the Novel Object Recognition (NOR) and Morris Water Maze (MWM) tasks. Golgi‐Cox Stain, Amyloid Beta immunohistochemistry, Western blots, SUnSET Assays, and a variety of other experiments were performed on the mice after sacrifice.ResultWe found that cognitive impairments displayed in aged Tg19959 and APP/PS1 mice were alleviated with treatment of A‐484954. Brain tissue from these mice also underwent high performance liquid chromatography (HPLC) to assess drug concentration. In addition, Golgi‐Cox stain was done to assess dendritic spine density and morphology. Spine analysis indicates a rescue in levels of mature spines in the CA1 region of the hippocampus in APP/PS1 mice treated with A‐484954.ConclusionTaken together, our results suggest that treatment with a eEF2K inhibitor, A‐484954, alleviates cognitive impairments and restores synaptic morphology in two mouse models of AD.

Memory impairments and synaptic deficits in Tg19959 and APP/PS1 AD model mice are alleviated by eEF2K inhibitor A‐484954

AbstractBackgroundMounting evidence indicates synaptic failure as an early and key event in Alzheimer’s Disease (AD) pathophysiology. Maintenance of long‐term memory and synaptic plasticity requires de novo protein synthesis. Phosphorylation of mRNA translation factor eukaryotic elongation factor 2 (eEF2) by its kinase eEF2K results in inhibition of general protein synthesis. Previous studies have shown elevated levels of eEF2 phosphorylation in post mortem AD human brain tissue and in AD mouse models. Here, we investigated whether suppression of eEF2 phosphorylation via eEF2K inhibitor A‐484954 can alleviate AD‐associated synaptic failure and memory impairments.MethodAged Tg19959 mice (6‐9 months), APP/PS1 (12‐16 months) and age‐matched controls were treated with a subcutaneous pellet containing A‐484954 or vehicle. The pellet continuously releases treatment over 30 days. Starting two weeks after pellet placement, the mice underwent cognitive assessment via the Novel Object Recognition (NOR) and Morris Water Maze (MWM) tasks. Golgi‐Cox Stain, Amyloid Beta immunohistochemistry, Western blots, SUnSET Assays, mass spectrometry (MS) proteomic analysis, and transmission electron microscopy (TEM) were also performed.ResultWe found that cognitive impairments displayed in aged Tg19959 and APP/PS1 mice were alleviated with treatment of A‐484954. Brain tissue from these mice also underwent high performance liquid chromatography (HPLC) to assess drug concentration in the brain. In addition, Golgi‐Cox stain was done to assess dendritic spine density and morphology. Spine analysis indicates a rescue in levels of mature spines in the CA1 region of the hippocampus in APP/PS1 mice treated with A‐484954. TEM analysis indicated a rescue in dendritic polyribosome assembly, post‐synaptic density (PSD) formation, and PSD length. Additionally, SUnSET assays revealed a rescue in translational capacity in the hippocampi of AD mice treated with A‐484954.ConclusionTaken together, our results suggest that treatment with a eEF2K inhibitor, A‐484954, alleviates cognitive impairments, restores synaptic morphology, and rescues translational capacity in mouse models of AD.

Suppression of the kinase for elongation factor 2 alleviates mGluR-LTD impairments in a mouse model of Alzheimer’s disease

Impaired mRNA translation (protein synthesis) is linked to Alzheimer’s disease (AD) pathophysiology. Recent studies revealed the role of increased phosphorylation of eukaryotic elongation factor 2 (eEF2) in AD-associated cognitive deficits. Phosphorylation of eEF2 (at the Thr56 site) by its only known kinase eEF2K leads to inhibition of general protein synthesis. AD is considered as a disease of “synaptic failure” characterized by impairments of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Deficiency of metabotropic glutamate receptor 5-dependent LTD (mGluR-LTD) is indicated in cognitive syndromes associated with various neurological disorders, including AD, but the molecular signaling mechanisms underlying the mGluR-LTD dysregulation in AD remain unclear. In this brief communication, we report genetic repression of eEF2K in aged APP/PS1 AD model mice prevented AD-associated hippocampal mGluR-LTD deficits. Using a pharmacological approach, we further observed that impairments of mGluR-LTD in APP/PS1 mice were rescued by treating hippocampal slices with a small molecule eEF2K antagonist NH125. Our findings, taken together, suggest a critical role of abnormal protein synthesis dysregulation at the elongation phase in AD-associated mGluR-LTD failure, thus providing insights into a mechanistic understanding of synaptic impairments in AD and other related dementia syndromes.

MTORC1 controls long-term memory retrieval

Understanding how stored information emerges is a main question in the neurobiology of memory that is now increasingly gaining attention. However, molecular events underlying this memory stage, including involvement of protein synthesis, are not well defined. Mammalian target of rapamycin complex 1 (mTORC1), a central regulator of protein synthesis, has been implicated in synaptic plasticity and is required for memory formation. Using inhibitory avoidance (IA), we evaluated the role of mTORC1 in memory retrieval. Infusion of a selective mTORC1 inhibitor, rapamycin, into the dorsal hippocampus 15 or 40 min but not 3 h before testing at 24 h reversibly disrupted memory expression even in animals that had already expressed IA memory. Emetine, a general protein synthesis inhibitor, provoked a similar impairment. mTORC1 inhibition did not interfere with short-term memory retrieval. When infused before test at 7 or 14 but not at 28 days after training, rapamycin impaired memory expression. mTORC1 blockade in retrosplenial cortex, another structure required for IA memory, also impaired memory retention. In addition, pretest intrahippocampal rapamycin infusion impaired object location memory retrieval. Our results support the idea that ongoing protein synthesis mediated by activation of mTORC1 pathway is necessary for long but not for short term memory.

Hydrogen sulfide modulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation status in the integrated stress-response pathway

Hydrogen sulfide (H2S) regulates various physiological processes, including neuronal activity, vascular tone, inflammation, and energy metabolism. Moreover, H2S elicits cytoprotective effects against stressors in various cellular models of injury. However, the mechanism of the signaling pathways mediating the cytoprotective functions of H2S is not well understood. We previously uncovered a heme-dependent metabolic switch for transient induction of H2S production in the trans-sulfuration pathway. Here, we demonstrate that increased endogenous H2S production or its exogenous administration modulates major components of the integrated stress response promoting a metabolic state primed for stress response. We show that H2S transiently increases phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) resulting in inhibition of general protein synthesis. The H2S-induced increase in eIF2α phosphorylation was mediated at least in part by inhibition of protein phosphatase-1 (PP1c) via persulfidation at Cys-127. Overexpression of a PP1c cysteine mutant (C127S-PP1c) abrogated the H2S effect on eIF2α phosphorylation. Our data support a model in which H2S exerts its cytoprotective effect on ISR signaling by inducing a transient adaptive reprogramming of global mRNA translation. Although a transient increase in endogenous H2S production provides cytoprotection, its chronic increase such as in cystathionine β-synthase deficiency may pose a problem.

Possible mechanisms involved in the vasorelaxant effect produced by clobenzorex in aortic segments of rats.

Clobenzorex is a metabolic precursor of amphetamine indicated for the treatment of obesity. Amphetamines have been involved with cardiovascular side effects such as hypertension and pulmonary arterial hypertension. The aim of the present study was to investigate whether the direct application of 10–9–10–5 M clobenzorex on isolated phenylephrine-precontracted rat aortic rings produces vascular effects, and if so, what mechanisms may be involved. Clobenzorex produced an immediate concentration-dependent vasorelaxant effect at the higher concentrations (10–7.5–10–5 M). The present outcome was not modified by 10–6 M atropine (an antagonist of muscarinic acetylcholine receptors), 3.1×10–7 M glibenclamide (an ATP-sensitive K+ channel blocker), 10–3 M 4-aminopyridine (4-AP; a voltage-activated K+ channel blocker), 10–5 M indomethacin (a prostaglandin synthesis inhibitor), 10–5 M clotrimazole (a cytochrome P450 inhibitor) or 10–5 M cycloheximide (a general protein synthesis inhibitor). Contrarily, the clobenzorex-induced vasorelaxation was significantly attenuated (P<0.05) by 10–5 M L-NAME (a direct inhibitor of nitric oxide synthase), 10–7 M ODQ (an inhibitor of nitric oxide-sensitive guanylyl cyclase), 10–6 M KT 5823 (an inhibitor of protein kinase G), 10–2 M TEA (a Ca2+-activated K+ channel blocker and non-specific voltage-activated K+ channel blocker) and 10–7 M apamin plus 10–7 M charybdotoxin (blockers of small- and large-conductance Ca2+-activated K+ channels, respectively), and was blocked by 8×10–2 M potassium (a high concentration) and removal of the vascular endothelium. These results suggest that the direct vasorelaxant effect by clobenzorex on phenylephrine-precontracted rat aortic rings involved stimulation of the NO/cGMP/PKG/Ca2+-activated K+ channel pathway.

Transcriptional analysis of ORF amv133 of Amsacta moorei entomopoxvirus.

The open reading frame (ORF) amv133 of Amsacta moorei entomopoxvirus, encodes a putative lipase gene. Its temporal expression pattern was characterized by RT-PCR and found to start at 6 h postinfection (h p.i.) and reach a maximum level at 48 h p.i. While the ORF has a late promoter motif, the inhibition of viral DNA synthesis by Ara-C failed to inhibit transcription, but a general inhibitor of protein synthesis prevented its transcription, indicating that amv133 is an intermediate gene. 5'-RACE analysis showed that transcription was initiated at position -77 relative to the translational start site. To determine the size of the promoter, several truncations were generated and cloned upstream of the firefly luciferase reporter gene. The resulting constructs were tested in a dual assay. A fragment that contained 115 bp relative to the transcription start site exhibited optimum promoter length.

IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth

The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

A novel counter‐selection method for markerless genetic modification in Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a photosynthetic organism capable of efficient harnessing of solar energy while capturing CO(2) from the environment. Methods to genetically alter its genomic DNA are essential for elucidating gene functions and are useful tools for metabolic engineering. In this study, a novel counter-selection method for the genetic alteration of Synechocystis was developed. This method utilizes the nickel inducible expression of mazF, a general protein synthesis inhibitor, as a counter-selection marker. Counter-selection is particularly useful because the engineered strain is free of any markers which make further genetic modification independent of available antibiotic resistance genes. The usability of this method was further demonstrated by altering genes at several loci in two variants of Synechocystis.

Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase Mζ synthesis and induce immediate preconditioning protection of rat hippocampal slices

Anaesthetic preconditioning occurs when a volatile anaesthetic, such as sevoflurane, is administered before a hypoxic or ischaemic insult; this has been shown to improve neuronal recovery after the insult. We found that sevoflurane-induced preconditioning in the rat hippocampal slice enhances the hypoxic hyperpolarization of CA1 pyramidal neurons, delays and attenuates their hypoxic depolarization, and increases the number of neurons that recover their resting and action potentials after hypoxia. These altered electrophysiological effects and the improved recovery corresponded with an increase in the amount of a constitutively active, atypical protein kinase C isoform found in brain, protein kinase M zeta (PKMζ). A selective inhibitor of this kinase, zeta inhibitory peptide (ZIP), blocked the increase in the total amount of PKMζ protein and the amount of the activated form of this kinase, phospho-PKMζ (p-PKMζ); it also blocked the altered electrophysiological effects and the improved recovery. We found that both cycloheximide, a general protein synthesis inhibitor, and rapamycin, a selective inhibitor of the mTOR pathway for regulating protein synthesis, blocked the increase in p-PKMζ, the electrophysiological changes, and the improved recovery due to sevoflurane-induced preconditioning. Glibenclamide, a KATP channel blocker, when present only during the hypoxia, prevented the enhanced hyperpolarization, the delayed and attenuated hypoxic depolarization, and the improved recovery following sevoflurane-induced preconditioning. To examine the function of persistent PKMζ and KATP channel activity after the preconditioning was established, we administered 4% sevoflurane for 30 min and then discontinued it for 30 min before 10 min of hypoxia. When either tolbutamide, a KATP channel blocker, or ZIP were administered at least 15 min after the washout of sevoflurane, there was little recovery compared with sevoflurane alone. Thus, continuous KATP channel and PKMζ activity are required to maintain preconditioning protection. We conclude that sevoflurane induces activation of the mTOR pathway, increasing the new protein synthesis of PKMζ, which is constitutively phosphorylated to its active form, leading to an increased KATP channel-induced hyperpolarizaton. This hyperpolarization delays and attenuates the hypoxic depolarization, improving the recovery of neurons following hypoxia. Thus, sevoflurane acts via a metabotropic pathway to improve recovery following hypoxia.

Adenine-containing codons enhance protein synthesis by promoting mRNA binding to ribosomal 30S subunits provided that specific tRNAs are not exhausted

Adenines downstream of the initiation codon promote protein synthesis; however, some adenine-containing codons (AGA, AGG and AUA) at early positions inhibit protein synthesis when cognate tRNA is exhausted. It has also been reported, although not convincingly, the presence of adenines enhancing mRNA binding to the ribosome. To understand these apparent inconsistencies we analyzed the effect of these codons in mRNA-ribosome binding strength, mRNA stability, the production of peptidyl-tRNA (pep-tRNA) and protein synthesis. Constructs harboring lacZ derivatives were obtained by site directed mutagenesis where tandems of GGG, AGG, AGA, ATA and AAA codons were inserted at codon positions 2–3 and 3–4. Codons containing more adenines, irrespective of being common or rare, (AAA, ATA and AGA) promoted a higher synthesis of β-galactosidase (β-gal) in comparison with those rich in guanines (GGG and AGG) in a wild type transcription-translation system. Full-length mRNAs were also detected when the adenine-rich constructs were expressed in wild type cells. Under conditions where the pool of tRNAs is readily exhausted (pep-tRNA hydrolase defective cells), the adenine-rich lacZ derivatives caused a stronger and general inhibition of protein synthesis and cell growth. With the exception of the ATA lacZ derivative, only plasmid constructs containing hungry codons generated pep-tRNA (AGA and to a lesser extent AGG) in Pth defective cells. Codons containing more adenines clearly promoted lacZ mRNA binding to 30S subunit. The GGG lacZ mRNA showed a moderate increase in binding when mRNA secondary structures were disrupted by heating mRNAs before the binding assay which agrees with the lacZ mRNA secondary structures predicted with MFOLD. Altogether, these results indicate that mRNA binding to ribosome plays a major role in the enhancement of translation by adenine-rich codons irrespective of codon usage. This effect is naturally expressed in wild type systems and depends on adenine content, in contrast to the inhibition caused after over-expressing the lacZ derivatives containing rare codons in Pth defective cells.

Rotavirus Infection Induces the Unfolded Protein Response of the Cell and Controls It through the Nonstructural Protein NSP3

The unfolded protein response (UPR) is a cellular mechanism that is triggered in order to cope with the stress caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). This response is initiated by the endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER kinase, which increase the expression of the genes involved in the folding and degradation processes and decrease the protein input into the ER by inhibiting translation. It has been shown that viruses both induce and manipulate the UPR in order to protect the host cells from an ER stress-mediated death, thus permitting the translation of viral proteins and the efficient replication of the virus. To understand the cellular events that occur during the rotavirus replication cycle, we examined the activation of the three UPR arms following infection, using luciferase reporters driven by promoters of the ER stress-responsive genes and real-time reverse transcription-PCR to determine the levels of the stress-induced mRNAs. Our findings indicated that during rotavirus infection two of the three arms of the UPR (IRE1 and ATF6) become activated; however, these pathways are interrupted at the translational level by the general inhibition of protein synthesis caused by NSP3. This response seems to be triggered by more than one viral protein synthesized during the replication of the virus, but not by the viral double-stranded RNA (dsRNA), since cells transfected with psoralen-inactivated virions, or with naked viral dsRNA, did not induce UPR.

The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite.

Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2(-) sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.

Ex Vivo Activity of Cardiac Glycosides in Acute Leukaemia

BackgroundDespite years of interest in the anti-cancerous effects of cardiac glycosides (CGs), and numerous studies in vitro and in animals, it has not yet been possible to utilize this potential clinically. Reports have demonstrated promising in vitro effects on different targets as well as a possible therapeutic index/selectivity in vitro and in experimental animals. Recently, however, general inhibition of protein synthesis was suggested as the main mechanism of the anti-cancerous effects of CGs. In addition, evidence of species differences of a magnitude sufficient to explain the results of many studies called for reconsideration of earlier results.Principal FindingsIn this report we identified primary B-precursor and T-ALL cells as being particularly susceptible to the cytotoxic effects of CGs. Digitoxin appeared most potent and IC50 values for several patient samples were at concentrations that may be achieved in the clinic. Significant protein synthesis inhibition at concentrations corresponding to IC50 was demonstrated in colorectal tumour cell lines moderately resistant to the cytotoxic effects of digoxin and digitoxin, but not in highly sensitive leukaemia cell lines.ConclusionIt is suggested that further investigation regarding CGs may be focused on diagnoses like T- and B-precursor ALL.

A Short Period of Mannitol Stress but Not LiCl Stress Led to Global Translational Repression in Plants

In plant cells, high salinity stress induces rapid inhibition of general protein synthesis. In this study, we found that treatment with mannitol, but not lithium stress, led to rapid global translational repression, suggesting that a rapid response at the level of translation might be induced by the osmotic but not the ionic components of salinity stress.

Effect of fatty acids on translational control of leukocytes infected with HIV‐1

During a virus infection, host cells suffer a general inhibition of protein synthesis. Initially, the viral RNAs activate the protein double‐stranded RNA‐dependent protein kinase (PKR). Active PKR phosphorylates the eukaryotic translation initiation factor 2 (EIF2) and impairs the general protein synthesis. However, the Tat protein of the HIV‐1 virus inhibits EIF2 phosphorylation and so the synthesis of the viral proteins occurs freely. Tat is phosphorylated by PKR and activates the elongation of viral RNAs. Results from our group indicate that the palmitic acid (PA) stimulates EIF2 phosphorylation in Jurkat cells probably by a process involving the protein PKR‐like endoplasmatic reticulum kinase (PERK). The PERK protein phosphorylates EIF2 in response to endoplasmic reticulum stress. Under a controlled pulse of PA the phosphorylation of EIF2 is reversible and does not induce a significant toxicity. Indeed, the pulse of PA impairs the cell general protein synthesis as reveled by polysome profile assay. In addition, evidence has been obtained that PA can modulate HIV‐1 infection.

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

Gelsolin plays an important role in the regulation of amyloid beta-protein fibrillogenesis. We report here that calcium ionophore A23187 induces the expression of cytoplasmic gelsolin (c-gelsolin), and that protein kinase C (PKC) is involved in the up-regulation of c-gelsolin. In the presence of calcium, both SH-SY5Y and HEK-293 cells upon treatment with A23187 showed an increase in c-gelsolin expression in a concentration-dependent manner. Calcium-mediated up-regulation of c-gelsolin was inhibited by cycloheximide (a general inhibitor of protein synthesis). When cells were pretreated with staurosporine (an inhibitor of a variety of protein kinases including PKC), the up-regulation of c-gelsolin induced by A23187 was inhibited. Calphostin C, an inhibitor of PKC, blocked the up-regulation of c-gelsolin induced by A23187, while inhibitors of mitogen-activated protein kinases had no effect on c-gelsolin expression. In addition, phorbol-12-myristate-13-acetate, an activator of PKC, up-regulated c-gelsolin expression. These results suggest that calcium mediates up-regulation of c-gelsolin in a PKC-dependent manner.

Distinct Differences in in Vitro Erythroid Proliferation and Differentiation, p53 and Apoptosis Pathways in Diamond-Blackfan Anemia in Conjunction with Depletion of RPS19, RPL5 and RPL11 mRNA.

Abstract 176Identification of mutations in a number of ribosomal genes including RPS19, RPL5 and RPL11 in Diamond-Blackfan anenia has established it as a disease of aberrant ribosome biogenesis. To date, it has not been possible to determine if a common cellular mechanism accounts for the erythroid defect in DBA in conjunction with various ribosomal gene mutations identified. To address this issue, we infected normal CD34+ cells from cord blood with specific short hairpin (sh) RNA against RPS19 (shRPS19C), RPL5 (shRPL5A, B, C) and RPL11 (shRPL11A, B, C) and determined their ability to undergo erythroid differentiation in a liquid culture system. Efficiency of each shRNA was verified at the RNA level in UT7 cell lines and CD34+. They all decreased dramatically mRNA expression of RPS19, RPL5 and RPL11 from 90% to 50% depending on the shRNA and the cells, except shRPL11B and C, which reduced RPL11 mRNA expression from 40% to 20%. Erythroid precursors after sh RNA infection were harvested at D7, D9, D11, D14 and D16 and analyzed for cell clonogenicity, erythroid cell differentiation (May Grünwald Giemsa or flow cytometry), apoptosis (Tunel assay and flow cytometry), and mRNA and protein expression. During in vitro erythroid differentiation, we noted a decrease in the extent of cell amplification, from D0 (CD34+ cells) to D7 (CFU-e/proerythroblasts), to D9 (basophilic erythroblasts), to D11, D14, D16 (orthophilic erythroblasts) for all the shRPS19C, shRPL5 (A, B, C) and shRPL11A infected cells compared to uninfected or scramble infected cells. The decrease in erythroid proliferation was less important (50%) in shRPS19 infected cells compared to shRPL5A,B,C and shRPL11A infected CD34+ cells (90–95%). In association with documented decrease in expression of RPS19 mRNA after shRPS19 CD34+ infection, we did not find either an alteration in erythroid differentiation or increased apoptosis at any stage of cell differentiation. In marked contrast, CD34+ cells infected with either shRPL5A,B,C or shRPL11A exhibited importantly a delayed erythroid differentiation with a marked increase in apoptosis. These findings have enabled us to document that while decreased cell proliferation is a feature of all CD34+ cells with decreased expression of different ribosomal proteins, defective differentiation and increased apoptosis of erythroid cells is a distinguishing feature of only CD34+ cells with decreased expression of RPL5 and RPL11.To obtain mechanistic insights into the observed differences in apoptotic phenotype, we analyzed p53 and apoptosis pathways of CD34+ following infection with shRPS19, shRPL5A-B-C, and shRPL11A. All the proteins tested: p53, p21, caspase 3, Bax, Noxa exhibited a decreased expression levels following infection with shRPL5A,B,C and shRPL11A, while exhibiting an increased expression at the mRNA level, suggesting a general inhibition of protein synthesis following RPL5 and RPL11 mRNA depletion. Strikingly, RPS19 depletion resulted increased levels of p53 and p21 expression at both mRNA and protein levels, while Bax and Noxa (proapoptotic) mRNA were as the same level compared to the controls. These different patterns of alteration in p53 and apoptotic protein levels can account for different apoptotic phenotype observed following depletion of RPS19 and of RPL5 and RPL11.In summary, we identified for the first time distinct erythroid proliferation and differentiation defects in conjunction with different ribosomal protein defects. These findings may have implications for future genotype-phenotype relationships in DBA patients. Disclosures:No relevant conflicts of interest to declare.

Induction of the unfolded protein response by calcineurin inhibitors: a double-edged sword in renal transplantation

The endoplasmic reticulum (ER) provides a unique environment for appropriate protein folding and assembly to produce functional, mature proteins. A number of pathophysiological insults cause accumulation of unfolded proteins in the ER, namely, ER stress. In response to ER stress, cells adapt themselves to the stress conditions via the unfolded protein response (UPR), leading to attenuation of translation, induction of ER chaperones and activation of ER-associated degradation (ERAD) to eliminate immature proteins. The UPR is involved in a diverse range of pathophysiological events [1,2], including renal diseases [3]. In response to ER stress, three major branches of the UPR are activated, as summarized in Figure 1A. Those include the RNA-dependent protein kinase-like ER kinase (PERK) pathway, the activating transcription factor 6 (ATF6) pathway and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathway. Activation of PERK causes phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which leads to general inhibition of protein synthesis. In response to ER stress, p90ATF6 transits to the Golgi where it is cleaved by the proteases S1P and S2P, yielding an active transcription factor p50ATF6. Similarly, activated IRE1 catalyses removal of a small intron from the mRNA of X-box binding protein 1 (XBP1). This splicing event creates translational frameshift in XBP1 mRNA to produce an active transcription factor. Active p50ATF6 and XBP1 subsequently bind to the ER stress response element (ERSE) and the UPR element (UPRE), leading to expression of target genes including ER chaperones and ERAD factors that degrade unfolded proteins. Cyclosporine A (CsA) and tacrolimus (FK506) are pivotal immunosuppressive agents to prevent allograft rejection in renal transplantation. Use of these immunosuppressants leads to significant reduction in the incidence of acute graft rejection and improvement in the survival of kidney transplants [4]. CsA binds to the cyclophilin family of molecules that have high affinity for calcineurin, a key protein phosphatase in the activation of T cells. FK506 is another calcineurin inhibitor, the mechanism of which is similar to that of CsA. It forms complexes with its cytosolic partner FK506-binding protein 12, and the complexes bind to calcineurin. By blocking calcineurin, CsA and FK506 inhibit phosphatase-controlled translocation of nuclear factor of activated T-cells (NF-AT) into the nucleus and prevent induction of cytokines and their receptors required for activation and proliferation of lymphocytes and other immune cells [5]. These agents are, therefore, regarded as inhibitors of immune cell function. However, several reports have also demonstrated that CsA and FK506 suppress activation of non-immune cells including keratinocytes, endothelial cells, mesangial cells, synovial fibroblasts and hepatocytes. For example, CsA inhibits mitogenesis of endothelial cells and growth factor-driven proliferation of keratinocytes [6,7]. CsA also attenuates induction of cytokines and their receptors in keratinocytes and expression of nitric oxide synthase in mesangial cells [8,9]. Similarly, other investigators reported that FK506 inhibits secretion of TNF-α in keratinocytes, production of matrix metalloproteinase 13 by rheumatoid synovial fibroblasts and generation of reactive oxygen species and nitric oxide in hepatocytes [10–12]. Currently, molecular mechanisms underlying the suppressive effects of CsA and FK506 on the non-lymphoid lineages are largely unknown. Recently, we found that in renal tubular cells, induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines is blunted by CsA and FK506 [13]. We identified that this suppressive effect is ascribed, at least in part, to induction of ER stress and consequent UPR. This phenomenon is observed not only in tubular cells but also in other non-immune cells including smooth muscle cells, preadipocytes, mesangial cells and endothelial cells. Indeed, administration with CsA in mice causes rapid induction of the UPR in several organs [13]. In tumour necrosis factor-α (TNF-α)-exposed cells, suppression of MCP-1 by CsA and FK506 is associated with the blunted activation of nuclear factor-κB (NF-κB), and the suppression of NF-κB is reproduced by other UPR inducers. CsA and FK506, as well as other UPR inducers, cause up-regulation