Nitric oxide induction of IRE1-α-dependent CREB phosphorylation in human glioma cells

In this study, the function of nitric oxide (NO) in endoplasmic reticulum (ER)-related cell death in human glioma cells was investigated. Treatment of human CRT-MG cells with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and thapsigargin, an ER stress inducer, increased cytosolic Ca²⁺ and caused apoptosis in a dose-dependent manner. Expression of the ER-associated molecules inositol-requiring enzyme 1 (IRE1)-α, p-eIF, and Ero1-α were also elevated in thapsigargin- or NO donor-treated cells. Furthermore, thapsigargin and SNAP treatment increased IRE1-α nuclease activity, induced IRE1-α/TRAF2 complex formation, and increased p-JNK1/2 levels, suggesting that NO activates the IRE1-α/TRAF2/JNK pathway in the ER. Expression of IRE1-α increased concomitantly with cAMP responsive element binding protein (CREB) phosphorylation. siRNA knock down of IRE1-α reduced phospho-CREB levels and abolished its nuclear translocation. The levels of phospho-CREB and IRE1-α increased with NO donor concentration, which resulted in cell death. IRE1-α and phospho-CREB levels in glioblastoma U87MG cells were higher than those in normal astrocytes in response to NO. In addition, treatment with the intracellular cytokine interleukin-1β induced cell death associated with NO and increased IRE1-α and p-CREB levels. These data reveal that intracellular NO affects IRE1-α-dependent CREB phosphorylation in human glioma cells. Therefore, an IRE1-α-dependent phospho-CREB signaling pathway responsive to NO/Ca²⁺ may play an important role in regulating ER-related cell death in glioma.     

Mephebrindole,a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2α/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells

The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3′-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3′-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.     

Strong induction of tyrosine phosphorylation,intracellular calcium,nuclear transcription factors and interferongamma,but weak induction of IL-2 in naïve T cells stimulated by bacterial superantigen

The outcome of T cell receptor (TCR) engagement is controlled by the differential recruitment of a variety of pathways, depending on the nature of the TCR ligand. Studies on superantigens (SAGs) were among the first describing such differential signaling; however, reported results are inconsistent. We took a quantitative approach to reinvestigate this question. Using nai;ve T cells from TCR transgenic mice, we found that compared to the antigenic peptide from pigeon cytochrome c, the SAG staphylococcal enterotoxin A very efficiently (100-2000-fold more sensitive on a weight basis) induced tyrosine kinase activity, intracellular calcium increase, and interferon (IFN)gamma production. Up-regulation of CD25 and CD69 and proliferation were less efficiently induced (20-30-fold more sensitive), and interleukin (IL)-2 production was induced least efficiently (only 2-fold more sensitive). This differential activation profile that varies with the activation event analyzed is discussed with respect to the propensity for SAG to induce anergy.     

The magic of four： induction of pluripotent stem cells from somatic cells by Oct4, Sox2, Myc and Klf4

The developmental process from a fertilized egg to agrown adult is programmed with remarkable accuracy.While the genetic information of the fertilized egg and itsdescendent somatic cells are the same, it is the selectiveexpressions of the same genome that give rise to the 200or so different cell types in an adult. The differentiatedstates of these adult cells are maintained epigenetically,presumably through the modification of chromatins and theassociated histones. In higher mammals, it was thought thatthe differentiation process is irreversible until the successfulcloning of Dolly [1]. By transferring a nucleus from a fullydifferentiated cell in the mammary gland, Wilmut and col-leagues were able to generate an exact replica of a highermammal, the Doily [1]. This work not only demonstratedthat the genome of differentiated cells can be reprogrammedinto an embryonic state and then to resume a full-fledgeddevelopmental process to generate a normal adult, but alsorejuvenated the field of animal cloning. The prospect thata somatic cell from a patient may be reprogrammed to the     

Brain-specific disruption of the eIF2α kinase PERK decreases ATF4 expression and impairs behavioral flexibility

Translational control depends on phosphorylation of eIF2α by PKR-like ER kinase (PERK). To examine the role of PERK in cognitive function, we selectively disrupted PERK expression in the adult mouse forebrain. In the prefrontal cortex (PFC) of PERK-deficient mice, eIF2α phosphorylation and ATF4 expression were diminished and were associated with enhanced behavioral perseveration, decreased prepulse inhibition, reduced fear extinction, and impaired behavioral flexibility. Treatment with the glycine transporter inhibitor SSR504734 normalized eIF2α phosphorylation, ATF4 expression, and behavioral flexibility in PERK-deficient mice. Moreover, the expression levels of PERK and ATF4 were reduced in the frontal cortex of human patients with schizophrenia. Together, our findings reveal that PERK plays a critical role in information processing and cognitive function and that modulation of eIF2α phosphorylation and ATF4 expression may represent an effective strategy for treating behavioral inflexibility associated with several neurological disorders such as schizophrenia.     

Neurturin, RET, GFRα-1 and GFRα-2, but not GFRα-3, mRNA are expressed in mice gonads

The gonads are known to produce numerous hormones and also neurotrophins and their receptors. Here we demonstrate expression of glial-cell-line-derived neurotrophic factor (GDNF) family ligands and related receptors in adult mice gonads by in situ hybridization. GDNF mRNA was expressed in the ovary, but was not detectable in testis. Neurturin (NTN), another ligand in this family, gave rise to strong mRNA hybridization signals in a mosaic pattern in the seminiferous tubules of the testis at stages IX-XII and I-II of the cycle. NTN mRNA signals were also found in uterus and the oviduct. In testis, the transducing receptor RET as well as GDNF receptor alpha-1 (GFR)alpha-1 and GFRalpha-2 were distributed in complementary and overlapping patterns, the former at stages XI-XII-I and the latter at stages VII and VIII. GFRalpha-3 could not be detected. Expression of these trophic molecules suggests involvement of GDNF family ligands and related receptor components in reproduction.     

Mnk mediates integrin α6β4-dependent eIF4E phosphorylation and translation of VEGF mRNA

It was previously shown that integrin α6β4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6β4 regulates the activity of eIF4E through the Ser/Thr kinase Mnk. Although a role for Mnk in various aspects of cancer progression has been established, a link between integrin and Mnk activity has not. Here we show that Mnk1 is a downstream effector of integrin α6β4 and mediates the α6β4 signaling, important for translational control. Integrin α6β4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6β4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6β4 level is high.     

Inhibition of eIF2α dephosphorylation enhances TRAIL-induced apoptosis in hepatoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inducer of cancer cell death that holds promise in cancer therapy. Cancer cells are more susceptible than normal cells to the cell-death-inducing effects of TRAIL. However, a variety of cancer cells are resistant to TRAIL through complex mechanisms. Here, we investigate the effects of inhibition of eukaryotic initiation factor 2 subunit α (eIF2α) dephosphorylation on TRAIL-induced apoptosis in hepatoma cells. Treatment of hepatoma cells with salubrinal, an inhibitor of eIF2α dephosphorylation, enhances TRAIL-induced eIF2α phosphorylation, CCAAT/enhancer-binding protein homologous protein (CHOP) expression and caspase activation. Salubrinal enhances TRAIL-induced apoptosis, which could be abrogated by caspase inhibitor. Overexpression of phosphomimetic eIF2α (S51D) enhances TRAIL-induced CHOP expression, caspase 7 and PARP cleavage and apoptosis. By contrast, overexpression of phosphodeficient eIF2α (S51A) abrogates the stimulation of TRAIL-induced apoptosis by salubrinal. Moreover, knockdown of growth arrest and DNA damage-inducible protein 34 (GADD34), which recruits protein phosphatase 1 to dephosphorylate eIF2α, enhances TRAIL-induced eIF2α phosphorylation, CHOP expression, caspase activation and apoptosis. Furthermore, the sensitization of hepatoma cells to TRAIL by salubrinal is dependent on CHOP. Knockdown of CHOP abrogates the stimulation of TRAIL-induced caspase activation and apoptosis by salubrinal. Combination of salubrinal and TRAIL leads to increased expression of Bim, a CHOP-regulated proapoptotic protein. Bim knockdown blunts the stimulatory effect of salubrinal on TRAIL-induced apoptosis. Collectively, these findings suggest that inhibition of eIF2α dephosphorylation may lead to synthetic lethality in TRAIL-treated hepatoma cells.