DNA Damage-inducible Protein Research Articles

Huwe1 interacts with Gadd45b under oxygen-glucose deprivation and reperfusion injury in primary Rat cortical neuronal cells.

BackgroundGrowth arrest and DNA-damage inducible protein 45 beta (Gadd45b) is serving as a neuronal activity sensor. Brain ischemia induces the expression of Gadd45b, which stimulates recovery after stroke and may play a protective role in cerebral ischemia. However, little is known of the molecular mechanisms of how Gadd45b expression regulated and the down-stream targets in brain ischemia. Here, using an oxygen-glucose deprivation and reperfusion (OGD/R) model, we identified Huwe1/Mule/ARF-BP1, a HECT domain containing ubiquitin ligase, involved in the control of Gadd45b protein level. In this study, we also investigated the role of Huwe1-Gadd45b mediated pathway in BDNF methylation.ResultsWe found that the depletion of Huwe1 by lentivirus shRNA mediated interference significantly increased the expression of Gadd45b and BDNF at 24 h after OGD. Moreover, treatment with Cycloheximide (CHX) inhibited endogenous expression of Gadd45b, and promoted expression of Gadd45b after co-treated with lentivirus shRNA-Huwe1. Inhibition of Gadd45b by lentivirus shRNA decreased the expression levels of brain derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein (p-CREB) pathway, while inhibition of Huwe1 increased the expression levels of BDNF and p-CREB. Moreover, shRNA-Huwe1 treatment decreased the methylation level of the fifth CpG islands (123 bp apart from BDNF IXa), while shRNA-Gadd45b treatment increased the methylation level of the forth CpG islands (105 bp apart from BDNF IXa).ConclusionsThese findings suggested that Huwe1 involved in the regulation of Gadd45b expression under OGD/R, providing a novel route for neurons following cerebral ischemia-reperfusion injury. It also indicated that the methylation of BDNF IXa was affected by Gadd45b as well as Huwe1 in the OGD/R model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0178-y) contains supplementary material, which is available to authorized users.

Efficacy of fine needle aspiration-yielded cell block combined with molecular markers in the diagnosis of thyroid nodules

Objective To study the clinical efficacy of cell block yielded by ultrasound-guided fine needle aspiration in the early diagnosis of thyroid cancer and differential diagnosis of thyroid follicular tumor. Methods Cytological smears were performed in 864 thyroid nodules by ultrasound-guided fine needle aspiration. Then the residual specimens were made as cell blocks. Diagnosis was made by observation of the organization structure of the cell block. Cell block sections from 78 nodules with diagnosis of follicular tumors were applied to the detection of immunocytochemical staining for minichromosome maintenance protein(MCM2), growth arrest and DNA damage-induced nuclear protein 153(GADD153), and Ki-67. Results Satisfaction rate of cytological smear combined with cell block specimen was 94.3%(815/864)and accuracy rate of the diagnosis was 94.0%(766/815), which were higher than in cytological smear group(P 0.05). Conclusions The combination of ultrasound-guided fine needle aspiration-yielded cell block and molecular markers has great significance in early diagnosis of thyroid cancer and differentially diagnosis of thyroid follicular tumor. (Chin J Endocrinol Metab, 2015, 31: 932-936) Key words: Thyroid; Fine needle aspiration; Cell block; Molecular marker; Follicular tumor

GADD34-deficient mice develop obesity, nonalcoholic fatty liver disease, hepatic carcinoma and insulin resistance.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity. DNA damage-inducible protein 34 (GADD34/Ppp1r15a), originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells, dephosphorylates several kinases that function in important signaling cascades, including dephosphorylation of eIF2α. We examined the effects of GADD34 on natural life span by using GADD34-deficient mice. Here we observed for the first time that with age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma, and insulin resistance. We found that myofibroblasts and immune cells infiltrated the portal veins of aged GADD34-deficient mouse livers. A high-fat diet (HFD) induced a higher level of steatosis in young GADD34-deficient mice compared with WT mice. Differentiation into fat is dependent on insulin signaling. Insulin signaling in young GADD34-deficient mice was higher than that in WT mice, which explained the higher fat differentiation of mouse embryonic fibroblasts (MEFs) observed in GADD34-deficient mice. Through aging or a HFD, insulin signaling in GADD34-deficient liver converted to be down regulated compared with WT mice. We found that a HFD or palmitate treatment converted insulin signaling by up-regulating TNF-α and JNK.

Effects of growth arrest and DNA damage-inducible protein 34 (GADD34) on inflammation-induced colon cancer in mice.

Background:Growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a) is a family of GADD proteins that are induced by DNA damage. GADD34 protein has been suggested to regulate inflammation or host defense systems. However, the in vivo function of GADD34 in inflammation is still unclear. Long lasting inflammation, such as that seen in Crohn's disease and ulcerative colitis, is associated with a higher incidence of colorectal cancer (CRC).Methods:Using a colitis-associated cancer model, we analysed GADD34-deficient (KO) mice to study the effect of GADD34 on colitis and colorectal tumorigenesis.Results:We found a higher incidence of CRC in wild-type (WT) mice than in GADD34KO mice. Moreover, dextran sodium sulfate (DSS)-induced inflammatory responses were downregulated by GADD34 deficiency. The expression of pro-inflammatory mediators such as TNFα, IL-6, and iNOS/NOS2 was higher in the colons of WT mice than GADD34KO mice. IL-6 is known to activate STAT3 signalling in colonic epithelial cells and subsequently induced epithelial proliferation. We found that IL-6-STAT3 signalling and epithelial proliferation were higher in WT mice compared with GADD34KO mice.Conclusions:These results indicated that GADD34 upregulated pro-inflammatory mediator production leading to a higher tumour burden following azoxymethane (AOM)/DSS treatment.

An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x(1-2)Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

Upregulation of Programmed Death-1 and Its Ligand in Cardiac Injury Models: Interaction with GADD153.

PurposeProgrammed Death-1 (PD-1) and its ligand, PD-L1, are regulators of immune/ inflammatory mechanisms. We explored the potential involvement of PD-1/PD-L1 pathway in the inflammatory response and tissue damage in cardiac injury models.Experimental DesignIschemic-reperfused and cryoinjured hearts were processed for flow cytometry and immunohistochemical studies for determination of cardiac PD-1 and PD-L1 in the context of assessment of the growth arrest- and DNA damage-inducible protein 153 (GADD153) which regulates both inflammation and cell death. Further, we explored the potential ability of injured cardiac cells to influence proliferation of T lymphocytes.ResultsThe isolated ischemic-reperfused hearts displayed marked increases in expression of PD-1 and PD-L1 in cardiomyocytes; however, immunofluorescent studies indicate that PD-1 and PD-L1 are not primarily co-expressed on the same cardiomyocytes. Upregulation of PD-1/PD-L1 was associated with a) marked increases in GADD153 and interleukin (IL)-17 but a mild increase in IL-10 and b) disruption of mitochondrial membrane potential (ψm) as well as apoptotic and necrotic cell death. Importantly, while isotype matching treatment did not affect the aforementioned changes, treatment with the PD-L1 blocking antibody reversed those effects in association with marked cardioprotection. Further, ischemic-reperfused cardiac cells reduced proliferation of T lymphocytes, an effect partially reversed by PD-L1 antibody. Subsequent studies using the cryoinjury model of myocardial infarction revealed significant increases in PD-1, PD-L1, GADD153 and IL-17 positive cells in association with significant apoptosis/necrosis.ConclusionsThe data suggest that upregulation of PD-1/PD-L1 pathway in cardiac injury models mediates tissue damage likely through a paracrine mechanism. Importantly, inhibition of T cell proliferation by ischemic-reperfused cardiac cells is consistent with the negative immunoregulatory role of PD-1/PD-L1 pathway, likely reflecting an endogenous cardiac mechanism to curtail the deleterious impact of infiltrating immune cells to the damaged myocardium. The balance of these countervailing effects determines the extent of cardiac injury.

Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model.

Growth arrest and DNA damage-inducible protein (GADD34/Ppp1r15a) is induced by various stimuli including DNA damage and ER stress. DNA damage and oncogene activation, accompanied by tumor-specific DNA repair defects and a failure to stall the cell cycle, are early markers of hepatocellular carcinoma (HCC). However, whether GADD34 accounts for regulating HCC tumorigenesis remains elusive. Here, we demonstrated that GADD34 expression was upregulated in the liver of mice after exposure to a carcinogen, diethylnitrosamine (DEN). In both acute and chronic DEN treatment models, GADD34 deficiency not only decreased oncogene expression, but also reduced hepatic damage. Moreover, loss of GADD34 attenuated immune cell infiltration, pro-inflammatory cytokine expression and hepatic compensatory proliferation. Finally, GADD34-deficient mice showed impaired hepatocarcinogenesis. Thus, the process of DEN-induced HCC proceeded as follows. First, DEN treatment induced DNA damage in hepatocytes, resulting in elevated expression of GADD34 in the liver. The increased expression of GADD34 augmented hepatic necrosis followed by elevated expression of interleukin (IL)-1β and monocyte chemoattractant protein 1. This process promoted immune cell infiltration and Kupffer cell/macrophage activation followed by production of reactive oxygen species and pro-tumorigenic cytokines such as IL-6 and tumor necrosis factor-α. The pro-tumorigenic cytokines stimulated compensatory proliferation of surviving and mutant hepatocytes. Together with oncogene c-Myc expression, these processes led to HCC. Our results suggest therapeutic opportunities for HCC by targeting GADD34-related pathways.

MiR-146a and miR-370 coordinate enterovirus 71-induced cell apoptosis through targeting SOS1 and GADD45β.

Enterovirus 71 (EV71) is an emerging life-threatening pathogen particularly in the Asia-Pacific region. The major pathogenic feature in EV71-infected cells is apoptosis. However, which molecular mechanism mainly contributes to EV71-induced apoptosis is not investigated thoroughly. MicroRNAs (MiRNAs), the newly discovered molecules, govern a wide range of biological functions through post-transcriptional regulation including viral pathogenesis. Whether miRNAs and messenger RNAs (mRNAs) coordinate to trigger host cell apoptosis in EV71 infection was investigated in this study. We conducted an apoptosis-oriented approach using both mRNA and miRNA profiling and bioinformatic analysis. We identified two major apoptosis-associated signalling pathways, Bcl2 antagonist of cell death (BAD) phosphorylation and p53-dependent apoptosis pathways, in which Son of sevenless homolog 1 (SOS1) and Growth arrest and DNA damage-inducible protein 45β (GADD45β) were predicted as targets of miR-146a and miR-370 respectively. Luciferase reporter assays and Western blots demonstrated the negative regulation between miR-146a and SOS1 and between miR-370 and GADD45β. Silencing of miR-146a restored SOS1 expression and partially attenuated EV71 infection-induced apoptosis. Conversely, ectopic expression of miR-370 decreased virus infection-induced GADD45β expression and also diminished apoptosis. Finally, the transfection of antagomiR-146a and miR-370 contributed to attenuating EV71 infection-induced apoptosis. Herein we clearly demonstrate that EV71-induced cell apoptosis is partly governed by altered miRNAs.

Novel SNPs in the growth arrest and DNA damage-inducible protein 45 alpha gene (GADD45A) associated with meat quality traits in Berkshire pigs.

This study was conducted to evaluate the porcine gene GADD45A (growth arrest and DNA-damage-inducible protein 45 alpha) as a positional candidate controlling quantitative trait loci (QTL) for meat quality traits on chromosome 6 (SSC6). Four exons of the porcine GADD45A gene were defined from cDNA and BAC clone sequences. A total of 4 single nucleotide polymorphisms (SNPs) were identified in porcine GADD45A. The association of these SNPs (g.196A>G, g.392C>A, g.955T>C and g.3247A>T) with meat quality traits was evaluated in 678 Berkshire pigs. The genotype distribution of only one SNP (g.3247A>T) conformed to Hardy Weinberg equilibrium in the pig population analyzed in this study, and the other SNPs were not in Hardy-Weinberg equilibrium. All four SNPs were significantly associated with meat quality traits. Three SNPS (g.196A>G, g.392C>A, and g.955T>C) showed similar significant association patterns for drip loss, cooking loss, meat color (lightness; MC_L and yellowness; MC_B), shear force and water-holding capacity traits. By contrast, g.3247A>T had a different association pattern with other traits such as intramuscular fat content (IMF) and backfat thickness (BF), drip loss, MC_L, and moisture. These findings will provide useful information for genetic characterization or association studies in other pig populations. Additionally, these markers can potentially be applied in pig breeding programs to improve meat quality traits, including IMF and BF.

Altering endoplasmic reticulum stress in a model of blast-induced traumatic brain injury controls cellular fate and ameliorates neuropsychiatric symptoms.

Neuronal injury following blast-induced traumatic brain injury (bTBI) increases the risk for neuropsychiatric disorders, yet the pathophysiology remains poorly understood. Blood-brain-barrier (BBB) disruption, endoplasmic reticulum (ER) stress, and apoptosis have all been implicated in bTBI. Microvessel compromise is a primary effect of bTBI and is postulated to cause subcellular secondary effects such as ER stress. What remains unclear is how these secondary effects progress to personality disorders in humans exposed to head trauma. To investigate this we exposed male rats to a clinically relevant bTBI model we have recently developed. The study examined initial BBB disruption using Evan’s blue (EB), ER stress mechanisms, apoptosis and impulsive-like behavior measured with elevated plus maze (EPM). Large BBB openings were observed immediately following bTBI, and persisted for at least 6 h. Data showed increased mRNA abundance of stress response genes at 3 h, with subsequent increases in the ER stress markers C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein 34 (GADD34) at 24 h. Caspase-12 and Caspase-3 were both cleaved at 24 h following bTBI. The ER stress inhibitor, salubrinal (SAL), was administered (1 mg/kg i.p.) to investigate its effects on neuronal injury and impulsive-like behavior associated with bTBI. SAL reduced CHOP protein expression, and diminished Caspase-3 cleavage, suggesting apoptosis attenuation. Interestingly, SAL also ameliorated impulsive-like behavior indicative of head trauma. These results suggest SAL plays a role in apoptosis regulation and the pathology of chronic disease. These observations provide evidence that bTBI involves ER stress and that the unfolded protein response (UPR) is a promising molecular target for the attenuation of neuronal injury.

Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms

Ethnopharmacological relevanceThe root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant which has been employed as a panacea for more than 2,000 years in China. It has the actions of invigorating primordial qi, recovering pulse and desertion, engendering liquid, and calming spirit. The water extract of Panax ginseng (WEG) has been used to treat kinds of central nervous system disorders, such as depression, insomnia, Alzheimer׳s disease and Parkinson׳s disease. Our previous work has demonstrated that WEG possessed antidepressant-like activities in both acute and chronic stress models of depression. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of WEG on corticosterone-induced apoptosis. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of WEG. Materials and methodsThe PC12 cells were treated with 250μmol/L corticosterone in the absence or presence of WEG for 24h, then 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) detection, Hoechst33342 staining and TUNEL staining were investigated to confirm the neuroprotection of WEG. Then, mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca2+ ([Ca2+]i), reactive oxygen species (ROS) concentration, and the expression level of glucocorticoid receptor (GR), heat shock protein 90 (Hsp90), histone deactylase 6 (HDAC6), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible protein 153 (GADD153), X-box DNA-binding protein-1 (XBP-1), caspase-12, cytochrome C, inhibitor of caspase-activated deoxyribonuclease (ICAD), caspase-3 and caspase-9 were assessed by Western Blot analysis to understand the molecule mechanisms of neuroprotection of WEG. ResultsWEG partly reversed corticosterone-induced damage in PC12 cells, which increased cell viability, decreased LDH release, and attenuated corticosterone-induced apoptosis as compared with the corticosterone-treated group. Mechanistically, compared with the corticosterone-treated group, WEG strongly attenuated [Ca2+]i overload and ROS level, and restored mitochondrial function, including mPTP and MMP. Furthermore, WEG strongly up-regulated the expression of GR and HDAC6, and down-regulated the expression of Hsp90, cytochrome C, ICAD, caspase-3, caspase-9 as well as endoplasmic reticulum (ER) stress-related proteins, such as GADD153, GRP78, XBP-1, and caspase-12. ConclusionWEG possessed neuroprotection against corticosterone-induced damage in PC12 cells, and the underlying molecule mechanisms was depended on the intervening of HDAC6 and HSP90 of the GR-related function proteins, and subsequent restoration of ER and mitochondria functions.

Chemopreventive mechanism of polypeptides from Chlamy Farreri (PCF) against UVB-induced malignant transformation of HaCaT cells.

To investigate polypeptide from Chlamy Farreri (PCF)'s protective effect against skin cancer, we used a cellular model of ultraviolet B (UVB)-induced malignant transformation. The human keratinocyte cell line HaCaT was repeatly exposed to UVB (10 mJ/cm(2), 20 times) and malignant transformation was confirmed by Gimesa staining, cell cycle analysis and various assays [anchorage independent growth, matrix metalloproteinase-9 (MMP9) activity, plating efficiency]. The malignant transformation was found to be effectively prevented by PCF pretreatment (2.84mM for 2h prior to each UVB exposure). We investigated the mechanism of PCF-mediated action by determining its effect on DNA methylation status of the tumour suppressor genes [P16 and ras association domain family 1 A (RASSF1A)] in the UVB-transformed cells. Both genes were found to be hypermethylated by chronic UVB exposure. The expression levels of P16, RASSF1A, DNA methyltransferases (DNMTs) and DNA damage inducible protein a (GADD45a) were measured by reverse transcriptase-polymerase chain reaction and western blotting. While chronic UVB exposure was found to suppress the expression of P16 and RASSF1A, it enhanced the expression of DNMT3b. In the early phase of UVB-induced malignant transformation, the GADD45a expression was increased, however, it declined with a continued irradiation of the cells. The UVB-induced DNA hypermethylation of P16 and RASSF1A and subsequent gene silencing was reversed by PCF treatment. The inhibition of DNMTs expression suggested that PCF blocked DNA methylation and thereby the silencing of tumour suppressor genes. Furthermore, the PCF-mediated substantial increase in GADD45a expression indicated that PCF promoted demethylation of tumour suppressor genes via GADD45a induction.

Induction of GADD45α protects M17 neuroblastoma cells against MPP*.

Growth arrest and DNA-damage-inducible protein 45α (GADD45α) is an important member of the family of growth arrest and DNA damage-inducible (GADD) proteins. The expression patterns and possible roles of GADD45α in Parkinson's disease (PD) are so far less understood. In this study, we found that 1-methyl-4-phenylpyridinium (MPP+) treatment up-regulates the expression of GADD45α in both a time-dependent manner and a dose-dependent manner in human dopamine neuroblastoma M17 cells. The up-regulation of GADD45α was abolished by pretreatment with the c-Jun N-terminal kinases (JNK) inhibitor SP600125 but not the p38 specific inhibitor SB203580. Further study revealed that c-Jun silencing abolished the effects of MPP+ on the expression of GADD45α. Important, ChIP studies verified the ability of c-Jun to bind to the GADD45 promoter. In addition, we found that inhibition of GADD45α by small RNA interference exacerbates the impaired cell viability, LDH release, and apoptosis induced by MPP+. Correspondingly, silence of GADD45 exacerbated Caspase-3 activation induced by MPP+. These data suggested a neuroprotective effect of GADD45α against MPP+ neurotoxicity.

Comparative analysis of developmentally regulated expressions of Gadd45a, Gadd45b, and Gadd45g in the mouse and marmoset cerebral cortex

The cerebral cortex is an indispensable region that is involved in higher cognitive function in the mammalian brain, and is particularly evolved in the primate brain. It has been demonstrated that cortical areas are formed by both innate and activity-dependent mechanisms. However, it remains unknown what molecular changes induce cortical expansion and complexity during primate evolution. Active DNA methylation/demethylation is one of the epigenetic mechanisms that can modify gene expression via the methylation/demethylation of promoter regions. Three growth arrest and DNA damage-inducible small nuclear proteins, Gadd45 alpha, beta, and gamma, have been identified as regulators of methylation status. To understand the involvement of epigenetic factors in primate cortical evolution, we started by analyzing expression of these demethylation genes in the developing common marmoset (Callithrix jacchus) and mouse (Mus musculus) brain. In the marmoset brain, we found that cortical expression levels of Gadd45 alpha and gamma were reduced during development, whereas there was high expression of Gadd45 beta in some areas of the adult brain, including the prefrontal, temporal, posterior parietal and insula cortices, which are particularly expanded in greater primates and humans. Compared to the marmoset brain, there were no clear regional differences and constant or reduced Gadd45 expression was seen between juvenile and adult mouse brain. Double staining with a neuronal marker revealed that most Gadd45-expressing cells were NeuN-positive neurons. Thus, these results suggest the possibility that differential Gadd45 expression affects neurons, contributing cortical evolution and diversity.

GW25-e2334 Purification of Growth Arrest and DNA Damage-inducible Protein GADD45 Alpha and its Expression in Human Platelet and Human Myocardial Tissue

GW25-e2334 Purification of Growth Arrest and DNA Damage-inducible Protein GADD45 Alpha and its Expression in Human Platelet and Human Myocardial Tissue

A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response.

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response.

Influence of edaravone on growth arrest and DNA damage-inducible protein 34 expression following focal cerebral ischemia-reperfusion in rats

ABSTRACT Objective To investigate the influence of edaravone on the expression of growth arrest and DNA damage-inducible protein 34 (GADD34). Methods A total of 108 healthy male Sprague-Dawley rats were randomly divided into sham operation group, model group and edaravone group (36 cases for each group). Transient focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 h followed by reperfusion in Sprague-Dawley rats. Then, GADD34 expression was measured with immunohistochemistry at different time-points after reperfusion in the peri-infarct regions of all rats. Results The GADD34 expression was detected in the peri-infarct regions of rats 1 h after reperfusion, which reached its peak 24 h after reperfusion. And edaravone could significantly down-regulate the GADD34 expression. Conclusions Edaravon could down-regulate GADD34 expression, which suggests that edaravone may exert an important function in inhibiting endoplasmic reticulum stress reaction by scavenging free radicals in the upper stream.

Hes-1 SUMOylation by protein inhibitor of activated STAT1 enhances the suppressing effect of Hes-1 on GADD45α expression to increase cell survival.

BackgroundHairy and Enhancer of split 1 (Hes-1) is a transcriptional repressor that plays an important role in neuronal differentiation and development, but post-translational modifications of Hes-1 are much less known. In the present study, we aimed to investigate whether Hes-1 could be SUMO-modified and identify the candidate SUMO acceptors on Hes-1. We also wished to examine the role of the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) in SUMOylation of Hes-1 and the molecular mechanism of Hes-1 SUMOylation. Further, we aimed to identify the molecular target of Hes-1 and examine how Hes-1 SUMOylation affects its molecular target to affect cell survival.ResultsIn this study, by using HEK293T cells, we have found that Hes-1 could be SUMO-modified and Hes-1 SUMOylation was greatly enhanced by the SUMO E3 ligase PIAS1 at Lys8, Lys27 and Lys39. Furthermore, Hes-1 SUMOylation stabilized the Hes-1 protein and increased the transcriptional suppressing activity of Hes-1 on growth arrest and DNA damage-inducible protein alpha (GADD45α) expression. Overexpression of GADD45α increased, whereas knockdown of GADD45αα expression decreased cell apoptosis. In addition, H2O2 treatment increased the association between PIAS1 and Hes-1 and enhanced the SUMOylation of Hes-1 for endogenous protection. Overexpression of Hes-1 decreased H2O2-induced cell death, but this effect was blocked by transfection of the Hes-1 triple sumo-mutant (Hes-1 3KR). Overexpression of PIAS1 further facilitated the anti-apoptotic effect of Hes-1. Moreover, Hes-1 SUMOylation was independent of Hes-1 phosphorylation and vice versa.ConclusionsThe present results revealed, for the first time, that Hes-1 could be SUMO-modified by PIAS1 and GADD45α is a novel target of Hes-1. Further, Hes-1 SUMOylation mediates cell survival through enhanced suppression of GADD45α expression. These results revealed a novel role of Hes-1 in addition to its involvement in Notch signaling. They also implicate that SUMOylation could be an important posttranslational modification that regulates cell survival.

Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

SummaryMutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

Phosphatase holoenzyme PP1/GADD34 negatively regulates TLR response by inhibiting TAK1 serine 412 phosphorylation.

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-κB pathway and the production of TNF-α, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-β-activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R-triggered NF-κB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.