DNA Damage-inducible Transcript 4 Research Articles

Involvement of C/EBPβ-related signaling pathway in methamphetamine-induced neuronal autophagy and apoptosis

Methamphetamine (METH) is a widely abused illicit psychoactive drug. Our previous study has shown that CCAAT-enhancer binding protein β (C/EBPβ) is an important regulator in METH-induced neuronal autophagy and apoptosis. However, the detailed molecular mechanisms underlying this process remain poorly understood. Previous studies have demonstrated that DNA damage-inducible transcript 4 (DDIT4), Trib3 (tribbles pseudo kinase 3), alpha-synuclein (α-syn) are involved in METH-induced dopaminergic neurotoxicity. We hypothesized that C/EBPβ is involved in METH-induced DDIT4-mediated neuronal autophagy and Trib3-mediated neuronal apoptosis.We tested our hypothesis by examining the effects of silencing C/EBPβ, DDIT4, Trib3 or α-syn with small interfering ribonucleic acid (siRNA) on METH-induced autophagy and apoptosis in the human neuroblastoma SH-SY5Y cells. We also measured the levels of phosphorylated tuberous sclerosis complex 2 (TSC2) protein and Parkin protein level in SH-SY5Y cells. Furthermore, we demonstrated the effect of silencing C/EBPβ on METH-caused neurotoxicity in the striatum of rats by injecting LV-shC/EBPβ lentivirus using a stereotaxic positioning system. The results showed that METH exposure increased C/EBPβ, DDIT4 protein expression. Elevated DDIT4 expression raised up p-TSC2/TSC2 protein expression ratio, inhibited mTOR signaling pathway, activating cell autophagy. We also found that METH exposure increased the expression of Trib3, α-syn, decreased the Parkin protein expression. Lowering levels of Parkin raised up α-syn expression, which initiated mitochondrial apoptosis by down-regulating anti-apoptotic Bcl-2, followed by up-regulation of pro-apoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. These findings were supported by data showing METH-induced autophagy and apoptosis was significantly inhibited by silencing C/EBPβ, DDIT4, Trib3 or α-syn, or by Parkin over-expression. Based on the present data, a novel of mechanism on METH-induced cell toxicity is proposed, METH exposure increased C/EBPβ protein expression, triggered DDIT4/TSC2/mTOR signaling pathway, and evoked Trib3/Parkin/α-syn-related mitochondrial apoptotic signaling pathway. Collectively, these results suggest that C/EBPβ plays an important role in METH-triggered autophagy and apoptosis and it may be a potential target for therapeutics in METH-caused neurotoxicity.

The physiological mTOR complex 1 inhibitor DDIT4 mediates therapy resistance in glioblastoma

BackgroundDespite significant advances in the understanding of glioblastoma genetics and biology, survival is still poor. Hypoxia and nutrient depletion in the tumour microenvironment induce adaptive signalling and metabolic responses, which can influence sensitivity to therapeutic regimens. DNA damage-inducible transcript 4 (DDIT4) is a protein induced by hypoxia and in response to DNA stress. Mechanistically, DDIT4 inhibits mammalian target of rapamycin complex 1 (mTORC1) signalling by activation of the tuberous sclerosis 1/2 (TSC1/2) complex.MethodsUsing short hairpin RNA-mediated gene suppression as well as doxycycline-regulated gene induction, we developed a glioblastoma cell model to study effects of DDIT4 under conditions of the glioblastoma microenvironment and therapy.ResultsWe found an intact DDIT4-mTORC1 signalling axis in human glioblastoma cells that was inducible by hypoxia. Temozolomide and radiotherapy also induced DDIT4 and repressed mTORC1 activity in some glioblastoma cell lines. DDIT4 gene suppression sensitised glioma cells towards hypoxia-induced cell death, while DDIT4 overexpression protected them. Additionally, in clonogenic survival analyses, DDIT4 induction conferred protection from radiotherapy and temozolomide, while DDIT4 gene suppression sensitised cells.ConclusionsWe identified DDIT4 as a cell-intrinsic regulator for adaptive responses and therapy resistance in glioblastoma cells which may interfere with cell death induction by temozolomide, radiotherapy or hypoxia by inhibiting mTORC1 activity.

Cardamonin Induces Cell Cycle Arrest, Apoptosis and Alters Apoptosis Associated Gene Expression in WEHI-3 Mouse Leukemia Cells.

Cardamonin, the chalcone class, is one of the natural components from the spicy herbaceous plant (Alpinia conchigera Griff) and has anticancer activities in many human cancer cell lines. There is, however, no information to show that cardamonin induces cell apoptosis and alters apoptosis associated gene expressions in mouse leukemia cells. Thus, we investigated the effects of cardamonin on the apoptotic cell death and associated gene expression in mouse leukemia WEHI-3 cells in vitro. Results indicated that cardamonin decreased total viable cell number via induced cell morphological changes and apoptotic cell death in WEHI-3 cells that were assay by contrast-phase microscopy and flow cytometry examinations, respectively. The flow cytometry assay indicated that cardamonin increased reactive oxygen species (ROS) and Ca production, decreased the levels of mitochondrial membrane potential ( and increased caspase-3, -8 and -9 activities in WEHI-3 cells. Western blotting was performed to analyze expression of relevant pro- and anti-apoptotic proteins and results showed that cardamonin decreased anti-apoptotic protein of Bcl-2 but increased pro-apoptotic protein of Bax in WEHI-3 cells. Furthermore, cardamonin increased cytochrome c, AIF and Endo G release, increased GRP78, caspase-12 that were associated with ER stress and increased Fas, Fas-Ligand and FADD expression. Furthermore, cardamonin increased the gene expressions of DAP (death-associated protein), TMBIM4 transmembrane (BAX inhibitor motif containing 4), ATG5 (autophagy related 5) but decreased the gene expression of DDIT3 (DNA-damage inducible transcript 3), DDIT4 (DNA-damage-inducible transcript 4), BAG6 (BCL2-associated athanogene 6), BCL2L13 [BCL2-like 13 (apoptosis facilitator)] and BRAT1 (BRCA1-associated ATM activator 1) that are associated with apoptosis pathways. Based on those findings, we may suggest cardamonin induced apoptotic cell death through Fas and Fas-Ligand-, caspase- and mitochondria-dependently pathways and also affects the apoptotic gene expression in WEHI-3 cells in vitro.

DNA damage-inducible transcript 4 is an innate guardian for human squamous cell carcinoma and an molecular vector for anti-carcinoma effect of 1,25(OH)2 D3.

Cutaneous squamous cell carcinoma (SCC) is one of the most common non-melanoma skin cancers worldwide. While its exact tumorigenesis mechanisms is far from well-established and less satisfied therapeutic strategy can be clinically used nowadays. In this study, we intended to investigate the role of DNA damage-inducible transcript 4 (DDIT4) in human SCC. Firstly, we identified DDIT4 is significantly suppressed in human SCC tissue and cultured A431 cell line, and reduced DDIT4 accelerates keratinocytes proliferation but impedes the autophagy flux through mTORC1 pathway by affecting the downstream S6 Kinase1, 4E-BP1, Beclin1 and LC3 II/I. While 1,25(OH)2 D3 enhanced DDIT4 expression and activated autophagy and inhibit mTORC1 to take the effect of anti-proliferation and activating autophagy. Further, formation of direct vitamin D receptor (VDR)-DDIT4 transcription complex was verified by ChIP-qPCR, which showed the molecular mechanism of how 1,25(OH)2 D3 promotes DDIT4 transcription. Thirdly, xenograft tumor-bearing mice model treated by gradient concentrations of 1,25(OH)2 D3 revealed the obvious anti-carcinoma effect of 1,25(OH)2 D3 in vivo and DDIT4 acted the molecular vector of 1,25(OH)2 D3 through mTORC1. Lastly, elevated DDIT4 expression was verified in human actinic keratoses tissue, and chronic long-term ultraviolet (UV) irradiation on mouse disclosed UV could promote DDIT4 expression inside epidermis. Conclusively, our research suggested a novel molecular mechanism about the human SCC tumorigenesis and the pharmacological mechanism about how 1,25(OH)2 D3 take its anti-carcinoma role on human SCC, as well as a striking paradoxes that how UV irradiation plays the tumorigenesis effect but synchronously take a protective role in the early stage of SCC carcinogenesis.

DDIT4 promotes gastric cancer proliferation and tumorigenesis through the p53 and MAPK pathways

BackgroundGastric cancer (GC) is one of the most common malignancies worldwide, particularly in China. DNA damage-inducible transcript 4 (DDIT4) is a mammalian target of rapamycin inhibitor and is induced by various cellular stresses; however, its critical role in GC remains poorly understood. The present study aimed to investigate the potential relationship and the underlying mechanism between DDIT4 and GC development.MethodsWe used western blotting, real-time polymerase chain reaction, and immunohistochemical or immunofluorescence to determine DDIT4 expression in GC cells and tissues. High-content screening, cell counting kit-8 assays, colony formation, and in vivo tumorigenesis assays were performed to evaluate cell proliferation. Flow cytometry was used to investigate cell apoptosis and cell cycle distribution.ResultsDDIT4 was upregulated in GC cells and tissue. Furthermore, downregulating DDIT4 in GC cells inhibited proliferation both in vitro and in vivo and increased 5-fluorouracil-induced apoptosis and cell cycle arrest. In contrast, ectopic expression of DDIT4 in normal gastric epithelial cells promoted proliferation and attenuated chemosensitivity. Further analysis indicated that the mitogen-activated protein kinase and p53 signaling pathways were involved in the suppression of proliferation, and increased chemosensitivity upon DDIT4 downregulation.ConclusionDDIT4 promotes GC proliferation and tumorigenesis, providing new insights into the role of DDIT4 in the tumorigenesis of human GC.

DNA Damage Inducible Transcript 4 Gene: The Switch of the Metabolism as Potential Target in Cancer

DNA damage inducible transcript 4 (DDIT4) gene is expressed under stress situations turning off the metabolic activity triggered by the mammalian target of rapamycin (mTOR). Several in vitro and in vivo works have demonstrated the ability of DDIT4 to generate resistance to cancer therapy. The link between the metabolism suppression and aggressiveness features of cancer cells remains poorly understood since anti-mTOR agents who are part of the repertoire of drugs used for systemic treatment of cancer achieving variable results. Interestingly, the high DDIT4 expression is associated with worse outcomes compared to tumors with low DDIT4 expression, seen in a wide variety of solid and hematological tumors, which suggests the driver role of this gene and provide the basis to target it as part of a new therapeutic strategy. In this review, we highlight our current knowledge about the biology of DDIT4 and its role as a prognostic biomarker, encompassing the motives for the development of target drugs against DDIT4 as a better target than mTOR inhibitors.

DDIT4 and Associated lncDDIT4 Modulate Th17 Differentiation through the DDIT4/TSC/mTOR Pathway.

Inflammation that complicates many autoimmune diseases, such as multiple sclerosis (MS), has been correlated to abnormal differentiation of Th17 cells. However, the reasons that promote Th17 cell-driven autoimmunity are yet to be discovered. In this study, we sought evidence that DNA-damage-inducible transcript 4 (DDIT4) and its associated long noncoding RNA DDIT4 (lncDDIT4) inhibit Th17 cell differentiation. We recruited 36 patients. Six MS patients and five healthy volunteers (controls) contributed PBMCs as material for microarray analysis. Microarray assays of lncDDIT4 and DDIT4 RNA expression identified outstanding differences between MS and control subjects, which were verified with real-time quantitative PCR. We then interrupted the expression of lncDDIT4 and DDIT4 mRNA in MS patients' naive CD4+ T cells and observed the resulting changes in Th17 cells. The expression of lncDDIT4 and DDIT4 mRNA were higher both in PBMCs and CD4+ T cells of MS patients than in healthy controls. DDIT4 (2.79-fold upregulation) was then recognized as a candidate for the cis-regulated target of lncDDIT4 (4.32-fold upregulation). Isolation of naive CD4+ T cells revealed enhanced levels of lncDDIT4 and DDIT4 after stimulated with Th17-inducing cytokines, but not after Th1, Th2, or T regulatory cell induction. Overexpression of lncDDIT4 in naive CD4+ T cells inhibited IL-17 transcription through increased DDIT4 expression and decreased activation of the DDIT4/mTOR pathway. Consistently, silencing lncDDIT4 in naive CD4+ T cells enhanced Th17 differentiation through increased activation of the DDIT4/mTOR pathway. However, these results vanished when DDIT4 was silenced. This outcome suggests that lncDDIT4 regulates Th17 cell differentiation by directly targeting DDIT4.

Dysfunction of DNA damage-inducible transcript 4 in the decidua is relevant to the pathogenesis of preeclampsia.

Preeclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation and affects 3-5% of all human pregnancies worldwide. However, the pathogenesis of PE still remains poorly understood. A deficiency in decidualization is considered a contributing factor to the development of PE. The DNA damage inducible transcript 4 (DDIT4) gene encodes a protein whose main function is inhibiting mammalian target of rapamycin (mTOR) under stress, and several studies have demonstrated that its expression promotes tumor cell apoptosis. Our previous RNA-Seq results showed that DDIT4 is significantly decreased in the decidua of PE women. Here, we aimed to define the role of DDIT4 in human decidualization and its relationship with PE. The results indicated that DDIT4 was markedly decreased in the decidua of severe PE compared with those from uncomplicated pregnancies. The expression of DDIT4 in human endometrial stromal cell (hESC) line and primary hESCs was up-regulated during decidualization. Knockdown DDIT4 in hESCs and primary hESCs caused a significant reduction in the transcription of decidualization markers, insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). In addition, silencing DDIT4 caused up-regulated p-mTOR and p-p70s6k and reduced apoptosis, whereas rapamycin, an inhibitor of mTOR, reversed the result of apoptosis. Moreover, the expression of cleaved-caspase 3 in severe PE was significantly lower than that of uncomplicated pregnancies, which was unfavorable for trophoblast invasion. Our data suggest that DDIT4 is critical for normal decidualization and the apoptosis of decidual cells. DDIT4 deficiency is likely involved in the development of PE.

Downregulation of microRNA‑124‑3p suppresses the mTOR signaling pathway by targeting DDIT4 in males with major depressive disorder.

Recent investigations have suggested that microRNAs (miRNAs or miRs) are involved in several pathways that may contribute to the pathomechanism of major depressive disorder(MDD). Sex may not only act as a demographic factor in clinical practive, but may also play a vital role in the molecular heterogeneity of MDD. Although many molecular changes correlated with MDD are found in males, the molecular mechanisms of MDD remain poorly understood. The present study performed bioinformatics analysis to investigate the pathomechanism of MDD in males. The present study identified miR‑124‑3p as one of the most dysregulated miRNAs in MDD, with decreased expression in the post‑mortem BA44 brain area of male patients with MDD. In addition, miR‑124‑3p targets DNA damage‑inducible transcript4(DDIT4) and specificity protein1(SP1), a DDIT4 transcription factor, in the validated target module of the miRWalk 2.0database. This is concurrent with an increase in the expression level of DDIT4, which is an inhibitor of the mammalian target of rapamycin(mTOR) signaling pathway. It was also demonstrated that miR‑124‑3p expression was positively associated with mTOR signaling and this relationship was dependent on the tuberous sclerosis proteins 1/2 complex. Taken together, these results provided a novel insight on miR‑124‑3p involvement in the biological alterations of male patients with MDD and suggested that this miRNA may also serve as a male‑specific target for antidepressant treatment.

DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1α and mTOR signalling.

Stem cell fate decisions to remain quiescent, self-renew or differentiate are largely governed by the interplay between extracellular signals from the niche and the cell intrinsic signal cascades and transcriptional programs. Here we demonstrate that DNA Damage Inducible Transcript 4 (DDIT4) acts as a link between HIF1α and mTOR signalling and regulation of adult stem cell fate. Global gene expression analysis of mesenchymal stem cells (MSC) derived from single clones and live RNA cell sorting showed a direct correlation between DDIT4 and differentiation potentials of MSC. Loss and gain of function analysis demonstrated that DDIT4 activity is directly linked to regulation of mTOR signalling, expression of pluripotency genes and differentiation. Further we demonstrated that DDIT4 exert these effects down-stream to HIF1α. Our findings provide an insight in regulation of adult stem cells homeostasis by two major pathways with opposing functions to coordinate between states of self-renewal and differentiation.

Effects of DDIT4 in Methamphetamine-Induced Autophagy and Apoptosis in Dopaminergic Neurons.

Methamphetamine (METH) is an illicit psychoactive drug that can cause a variety of detrimental effects to the nervous system, especially dopaminergic pathways. We hypothesized that DNA damage-inducible transcript 4 (DDIT4) is involved in METH-induced dopaminergic neuronal autophagy and apoptosis. To test the hypothesis, we determined changes of DDIT4 protein expression and the level of autophagy in rat catecholaminergic PC12 cells and human dopaminergic SH-SY5Y cells, and in the hippocampus, prefrontal cortex, and striatum of Sprague Dawley rats exposed to METH. We also examined the effects of silencing DDIT4 expression on METH-induced dopaminergic neuronal autophagy using fluorescence microscopy and electron microscopy. Flow cytometry and Western blot were used to determine apoptosis and the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) after blocking DDIT4 expression in PC12 cells and SH-SY5Y cells with synthetic siRNA, as well as in the striatum of rats by injecting LV-shDDIT4 lentivirus using a stereotaxic positioning system. Our results showed that METH exposure increased DDIT4 expression that was accompanied with increased autophagy and apoptosis in PC12 cells (3mM) and SH-SY5Y cells (2mM), and in the hippocampus, prefrontal cortex, and striatum of rats. Inhibition of DDIT4 expression reduced METH-induced autophagy and apoptosis in vitro and in vivo. However, DDIT4-related effects were not observed at a low concentration of METH (1μM). These results suggest that DDIT4 plays an essential role in METH-induced dopaminergic neuronal autophagy and apoptosis at higher doses and may be a potential gene target for therapeutics in high-dose METH-induced neurotoxicity.

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes.

1, 25(OH)2D3 protects β cell against high glucose-induced apoptosis through mTOR suppressing

Diabetes mellitus is a leading cause of death and disability worldwide, which presents a serious public health crisis in China nowadays. It has been well recognized that excessive β-cell apoptosis is the key pathogenesis of diabetes, of which the mammalian target of rapamycin (mTOR) serves as the critical signaling pathway. Emerging evidence indicates that vitamin D deficiency acts as a potential risk factor for diabetes. The present study aims to test the hypothesis that 1 alpha, 25-dihydroxyvitamin D(3) [1, 25(OH)2D3] can inhibit β-cell apoptosis via the suppression of mTOR signaling pathway. β-cells (INS-1) were cultured in the context of normal glucose or high glucose media with or without 1, 25(OH)2D3 treatment. β-cell apoptosis was evaluated by inverted fluorescence microscope, flow cytometry and electron microscope, respectively. Quantitative RT-PCR and Western blotting were performed to assess the possible perturbations in mTOR signaling pathway. High glucose significantly increased β-cell apoptosis. Of importance, RT-PCR and Western blotting demonstrated that high glucose inhibited DNA-damage-inducible transcript 4 (DDIT4) and TSC1/TSC2, up-regulated Rheb/mTOR/p70S6K and enhanced expression of the apoptosis regulating proteins, such as phospho-Bcl-2, cytochrome C and cleaved caspase. Interestingly, 1, 25(OH)2D3 treatment reversed high glucose induced pathological changes in mTOR signaling pathway, restored expression of DDIT4 and TSC1/TSC2, blocked aberrant up-regulation of Rheb/mTOR/p70S6K and the apoptosis regulating proteins, and effectively inhibited β-cell apoptosis. Therefore, 1, 25(OH)2D3 treatment can effectively protects β cell against high glucose-induced apoptosis mainly via the suppression of mTOR signaling pathway, which may be considered as a potential therapy for patients with diabetes.

Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling.

Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed silymarin suppression of glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism. Anti-inflammatory effects arose with prolonged (i.e., 24 h) silymarin exposure, with suppression of multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB) and forkhead box O (FOXO). Studies with murine knock out cells revealed that silymarin inhibition of both mTOR and NF-κB was partially AMPK dependent, whereas silymarin inhibition of mTOR required DDIT4. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Thus, natural products activate stress and repair responses that culminate in an anti-inflammatory cellular phenotype. Natural products like silymarin may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation.

Transcriptional co-repressor function of the hippo pathway transducers YAP and TAZ.

YAP (yes-associated protein) and TAZ are oncogenic transcriptional co-activators downstream of the Hippo tumor-suppressor pathway. However, whether YAP and/or TAZ (YAP/TAZ) engage in transcriptional co-repression remains relatively unexplored. Here, we directly demonstrated that YAP/TAZ represses numerous target genes, including tumor-suppressor genes such as DDIT4 (DNA-damage-inducible transcript 4) and Trail (TNF-related apoptosis-inducing ligand). Mechanistically, the repressor function of YAP/TAZ requires TEAD (TEA domain) transcription factors. A YAP/TAZ-TEAD complex recruits the NuRD complex to deacetylate histones and alters nucleosome occupancy at target genes. Functionally, repression of DDIT4 and Trail by YAP/TAZ is required for mTORC1 (mechanistic target of rapamycin complex 1) activation and cell survival, respectively. Our demonstration of the transcriptional co-repressor activity of YAP/TAZ opens a new avenue for understanding the Hippo signaling pathway.

Stress responses to short-term intensified and reduced training in competitive weightlifters.

We sought to identify and evaluate the tolerance to, and consequences of, short-term variations in training load in competitive weightlifters. Seven international-level lifters performed 1 week of initial training followed by 2 weeks of intensified (INT: +100%, 36.5 ± 11.3 × 10(3) kg/week) and 1 week of subsequently reduced (RED: -25%) training within their annual program. After INT, but not RED, 90 min of weightlifting increased mRNA levels of chemokine (C-C motif) ligand 4 (CCL4), chemokine (C-X-C motif) receptor 4 (CXCR4) and cellular stress-associated DNA-damage-inducible transcript 4 (DDIT4) in peripheral blood mononuclear cells by 40-240%. Resting- and weightlifting-induced changes in plasma protein carbonyls, indicative of oxidative stress, but not pro-inflammatory CCL4 concentrations differed between INT and RED. Symptoms of stress (Daily Analysis of Life Demands of Athletes questionnaire) were reported as worse than normal more frequently during INT and RED than initial training. Global (negative) mood state increased during INT and declined during RED. Maximal snatch (-4.3 ± 3.7%) and vertical jump (-7.2 ± 6.5%), but not clean and jerk, were reduced after INT and restored after RED. Chemokine signaling may thus be part of the stress response to intense weightlifting and short-term reductions in training load support recovery from periodic INT training in weightlifters.

Quantitative Lys-ϵ-Gly-Gly (diGly) Proteomics Coupled with Inducible RNAi Reveals Ubiquitin-mediated Proteolysis of DNA Damage-inducible Transcript 4 (DDIT4) by the E3 Ligase HUWE1

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.

REDD1/DDIT4-Independent mTORC1 Inhibition and Apoptosis by Glucocorticoids in Thymocytes

Glucocorticoids induce apoptosis in lymphocytes and are commonly used to treat hematologic malignancies. However, they are also associated with significant adverse effects and their molecular mechanism of action is not fully understood. Glucocorticoid treatment induces expression of the mTORC1 inhibitor Regulated in Development and DNA Damage Response 1 (REDD1), also known as DNA-Damage Inducible Transcript 4 (DDIT4), and mTORC1 inhibition may distinguish glucocorticoid-sensitive from glucocorticoid-resistant acute lymphoblastic leukemia (ALL). Interestingly, REDD1 induction was impaired in glucocorticoid-resistant ALL cells and inhibition of mTORC1 using rapamycin restored glucocorticoid sensitivity. These data suggest that REDD1 may be essential for the response of ALL cells to glucocorticoids. To further investigate the role of REDD1, we evaluated the effects of glucocorticoids on primary thymocytes from wild-type and REDD1-deficient mice. Glucocorticoid-mediated apoptosis was blocked by a glucocorticoid receptor antagonist and by an inhibitor of transcription, which interfered with REDD1 induction and mTORC1 inhibition. However, REDD1 ablation had no effect on glucocorticoid-induced mTORC1 inhibition and apoptosis in thymocytes ex vivo. Overall, these data not only demonstrate the contextual differences of downstream signaling following glucocorticoid treatment but also provide a better mechanistic understanding of the role of REDD1. These molecular findings underlying glucocorticoid action and the role of REDD1 are fundamental for the design of novel, more efficacious, and less toxic analogs. Mol Cancer Res; 12(6); 867-77. ©2014 AACR.

Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever

Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor β (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.

DDIT4 (DNA-damage-inducible transcript 4)

Review on DDIT4 (DNA-damage-inducible transcript 4), with data on DNA, on the protein encoded, and where the gene is implicated.