Targeting of EIF4EBP1 by miR-99a-3p affects the functions of B lymphocytes via autophagy and aggravates SLE disease progression

Excessive activation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNAs is a research hotspot. In this study, second-generation high-throughput sequencing revealed a reduction in miR-99a-3p expression in patients with SLE; however, the specific mechanism underlying this phenomenon remains unclear. After transfection with an miR-99a-3p agomir, the proliferation of Ball-1 cells decreased and the levels of their apoptosis increased. The opposite effects were observed in cells transfected with the miR-99a-3p antagomir. Luciferase reporter assay indicated that miR-99a-3p directly targeted EIF4EBP1. Rescue experiments confirmed the proposed interaction between miR-99a-3p and EIF4EBP1. In vitro, in vivo and clinical investigations further confirmed that the miR-99a-3p agomir reduced the expression of EIF4EBP1, LC3B and LAMP-2A. In the in vivo experiments, serum levels of anti-nuclear antibodies, double-stranded DNA, IgE, IgM, IL-6, IL-10 and B lymphocyte stimulator were higher in mice from the antagomir group than those in mice from the MRL/lpr group. Furthermore, the protein and mRNA levels of EIF4EBP1, LC3B and LAMP-2A, the intensity of immunohistochemical staining of EIF4EBP1, LC3B and LAMP-2A, the urinary protein levels, and the C3 immunofluorescence deposition increased in mice from the antagomir group. The upregulation of miR-99a-3p expression protected B cells from EIF4EBP1-mediated autophagy, whilst the downregulation of miR-99a-3p expression induced autophagy via the EIF4EBP1-mediated regulation of the autophagy signalling pathway in B cells isolated from individuals with SLE. Based on these results, miR-99a-3p and EIF4EBP1 may be considered potential targets for SLE treatment.     

SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-κB/STAT3 complex to its promoter in malignant lymphoid cells

We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies.     

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

Background

Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.

Methodology/Principal Findings

Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn''s disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.

Conclusions/Significance

Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.     

Hepcidin and hemojuvelin gene expression in rat liver damage: in vivo and in vitro studies

In this work, we used two rat models, partial hepatectomy (PH) and CCl(4) administration, to study the changes in iron pathways in response to hepatic damage. Liver injury induced changes in the hepatic gene expression of hepcidin, hemojuvelin (Hjv), several other proteins of iron metabolism, and several cytokines such as IL-1beta, IL-6, TNF-alpha, and IFN-gamma. Hepcidin gene expression was upregulated between 4 and 8 h with a maximum up to 16 h after surgery. However, Hjv gene expression was downregulated at the same time. An early upregulation of hepcidin (3 h) and downregulation of Hjv gene expression was found after CCl(4) administration. Transferrin receptor 1 and ferritin H gene expression was upregulated, whereas ferroportin 1 gene expression was downregulated. Hepatic IL-6 gene expression was upregulated early after PH and reached maximum 8 h after the PH. In CCl(4)-induced liver injury, IL-6, IL-1beta, TNF-alpha, and IFN-gamma upregulation were found at the maximum 12 h after the administration of the toxin. Treatment of isolated rat hepatocytes with IL-6 and, to a lesser extent, with IL-1beta but not with TNF-alpha or IFN-gamma dose dependently upregulated hepcidin and downregulated Hjv gene expression. In hepatic damage, changes of the hepatic gene expression of the main proteins involved in iron metabolism may be induced by locally synthesized mediators.     

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.     

EIF4EBP1 Overexpression Is Associated with Poor Survival and Disease Progression in Patients with Hepatocellular Carcinoma

ObjectiveEIF4EBP1 acts as a crucial effector in mTOR signaling pathway. Studies have suggested that EIF4EBP1 plays a critical role in carcinogenesis. However, the clinical significance and biological role of EIF4EBP1 in hepatocellular carcinoma (HCC) have not been elucidated. Therefore, we aimed to investigate the clinical significance of EIF4EBP1 in HCC.MethodsTotal 128 cases of HCCs were included in this study. EIF4EBP1 expression in HCC tissues was detected by qRT-PCR, Western blot and immunohistochemistry, respectively. Then the relationships between EIF4EBP1 expression and clinical features as well as survival were analyzed.ResultsThe expression level of EIF4EBP1 mRNA is significantly higher in 60% (24/40) of fresh HCC tissues than that in the matched adjacent nontumor liver (NCL) tissues (P = 0.044). Similarly, EIF4EBP1 protein is notably upregulated in 8 HCC tissues (randomly selected from the 40 HCCs) measured by Western blot and is significantly increased in another 88 paraffin-embedded HCCs (53%, 47/88) by immunohistochemistry compared with the matched NCLs (P < 0.001). EIF4EBP1 protein expression in HCC tissues is significantly correlated with serum AFP (P = 0.003) and marginally significantly associated with pathological grade (P = 0.085), tumor number (P = 0.084), tumor embolus (P = 0.084) and capsulation (P = 0.073). Patients with higher EIF4EBP1 protein expression have a much worse 5-year overall survival (40.3% vs 73.6%) and 5-year disease-free survival (33.0% vs 49.0%) than those with low expression. Furthermore, Cox regression analysis shows that EIF4EBP1 protein is an independent prognostic factor for overall survival (HR, 2.285; 95% CI, 1.154–4.527; P = 0.018) and disease-free survival (HR, 1.901; 95% CI, 1.067–3.386; P = 0.029) in HCC patients.ConclusionsOur results demonstrate for the first time that EIF4EBP1 mRNA and protein are markedly up-regulated in HCC tissues, and the protein overexpression is significantly associated with poor survival and progression, which provide a potential new prognostic marker and therapeutic target for HCC patients.     

Select nutrients in the ovine uterine lumen. IX. Differential effects of arginine, leucine, glutamine, and glucose on interferon tau, ornithine decarboxylase, and nitric oxide synthase in the ovine conceptus

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.     

Curcumin-induced GADD153 upregulation: modulation by glutathione

As we reported previously, GADD153 is upregulated in colon cancer cells exposed to curcumin. In the present study, we ascertained the involvement of glutathione and certain sulfhydryl enzymes associated with signal transduction in mediating the effect of curcumin on GADD153. Curcumin-induced GADD153 gene upregulation was attenuated by reduced glutathione (GSH) or N-acetylcysteine (NAC) and potentiated by the glutathione synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO). Additionally, GSH and NAC decreased the intracellular content of curcumin. Conversely, curcumin decreased intracellular glutathione and also increased the formation of reactive oxygen species (ROS) in cells, but either GSH or NAC prevented both of these effects of curcumin. In affecting the thiol redox status, curcumin caused activation of certain sulfhydryl enzymes involved in signal transduction linked to GADD153 expression. Curcumin increased the expression of the phosphorylated forms of PTK, PDK1, and PKC-delta, which was attenuated by either GSH or NAC and potentiated by BSO. Furthermore, selective inhibitors of PI3K and PKC-delta attenuated curcumin-induced GADD153 upregulation. Collectively, these findings suggest that a regulatory thiol redox-sensitive signaling cascade exists in the molecular pathway leading to induction of GADD153 expression as caused by curcumin.     

GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis

The main role of growth arrest and DNA damage-inducible (GADD) genes is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The goal of this study was to examine the expression of GADD genes in primary melanomas with respect to prognosis. GADD34 was found in 73% of the primary melanomas investigated. GADD45 and GADD153 were positive in 60% and 80% of primary melanomas, respectively. Cox regression demonstrated that only GADD153 had any independent prognostic impact. We therefore decided to establish a PCR assay for detection of GADD153 in paraffin-embedded tissue. GADD153 deletion was found in 3/26 melanomas. None of the 3 cases with GADD153 deletion showed any expression of GADD153. Sequencing analysis detected polymorphism T-C at amino acid position 10 in 6/23 melanomas. In 6 cases with GADD153 polymorphism, GADD153 expression was found in 2 melanomas with a maximum GADD153 index of 10%. We postulate that the GADD gene family plays an important role in melanoma progression.