Translational initiation regulated by ATM in dendritic cells development.

Ataxia telangiectasia mutated (ATM) protein has been implicated in multiple pathways such as DNA repair, cell cycle checkpoint, cell growth, development, and stem cell renewal. In this study, we demonstrate evidence that ATM is involved in granulocyte macrophage colony-stimulating factor (GM-CSF)-induced dendritic cell (DC) development from bone marrow (BM) cells. Inactivation of ATM protein results in decreased BM proliferation, leading to reduced DC development and their activity for T cell activation. Expression of Jak2, STAT5, and mTOR is suppressed in both wild-type and ATM-null BM prior to GM-CSF stimulation. Activation of those proteins is delayed and prolonged hypophosphorylation of 4EBP1 is observed in ATM-null BM when treated with GM-CSF, although Erk and p38 are similarly expressed and activated in both wild-type and ATM-null BM cell types. Akt is also suppressed in wild-type BM, and transduction of constitutively active Akt or STAT5 in ATM-null BM restores DC development. Together, these results illustrate that ATM deficiency causes impaired initiation of protein translation in BM, leading to immature development of DC.

DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway.

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage.

Phosphorylation of SMC1 by ATR is required for desferrioxamine (DFO)-induced apoptosis.

DNA damage signaling pathways are initiated in response to chemical reagents and radiation damage, as well as in response to hypoxia. It is implicated that structural maintenance of chromosomes 1 (SMC1) is not only a component of the cohesion complex but also facilitates the activation of DNA damage checkpoint proteins. Here, we studied the mechanism of DNA damage checkpoint activated by ATR-SMC1 pathway when cells are treated with desferrioxamine (DFO), a hypoxia-mimetic reagent. We show that DFO treatment induces phosphorylation of SMC1 at Ser966, NBS1 at Ser343, Chk1 at Ser317, Chk2 at Thr68, and p53 at Ser15. Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs. DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells. Expression of SMC1 S966A in which Ser966 is substituted to Ala attenuates apoptosis and phosphorylation of Chk1 at Ser317 after DFO treatment, although levels of HIF1α are not significantly changed. These results suggest that DFO induces apoptosis through the ATR-SMC1 arm of the pathway.

IFN-gamma and IFN-alpha posttranscriptionally down-regulate the IL-4-induced IL-4 receptor gene expression.

As Th1 and Th2 cytokines, IFN-gamma/alpha and IL-4 counterregulate diverse immune functions. In particular, IFN-gamma and IFN-alpha have been reported to markedly suppress the IL-4-induced IgE production and type II IgE receptor (FcepsilonRII/CD23) expression. Because modulation of IL-4R may be an important mechanism in the regulation of IL-4 response, we have investigated the effect of IFN-gamma/alpha on IL-4R expression and signal transduction mechanisms involved in this process. In human mononuclear cells and B cells isolated from tonsil or peripheral blood, IL-4 up-regulates IL-4R(alpha) expression at surface protein and mRNA levels, and the IL-4-induced IL-4R(alpha) is significantly down-regulated by both IFN-gamma and IFN-alpha to a similar extent. The inhibitory effects of IFN-gamma/alpha on the IL-4R mRNA expression require a lag period of about 8 h, and are sensitive to cycloheximide treatment, which suggests that the suppressive effect of IFNs on IL-4R gene expression is a secondary response requiring de novo synthesis of IFN-induced factors. Under such conditions that the inhibitory effects of IFNs are observed, IFNs do not affect the IL-4-induced STAT6 activation and IL-4R transcription, as analyzed by EMSA and nuclear run-on assays, respectively. Subsequently, mRNA stability studies have indicated that the action of IFN-gamma/alpha is primarily mediated by an accelerated decay of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in the case of the IL-4-induced FcepsilonRII regulation, posttranscriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which type I and II IFNs antagonize the IL-4 response in human immune cells.

Interferon-gamma-induced factor binding to the interleukin-4-responsive element of CD23b promoter in human tonsillar mononuclear cells: role in transient up-regulation of the interleukin-4-induced CD23b mRNA.

Stimulation of human tonsillar mononuclear cells with interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) rapidly induced the activation of distinct nuclear factors with different mobilities, both of which bind the IL-4 response element (IL-4RE) of CD23b promoter as examined by electrophoretic mobility shift assays (EMSA). Co-treatment of IL-4 and IFN-gamma induced, in addition to the two distinct complexes, a new complex with an intermediate mobility. The IL-4-induced complex reacted with anti-STAT (signal transducers and activators of transcription) 6, resulting in a supershift whereas the formation of the IFN-gamma-induced complex was inhibited by anti-STAT 1. The intermediate complex appeared to react with both anti-STAT 6 and anti-STAT 1. Although IFN-gamma alone did not induce CD23 mRNA transcription, Northern blot analysis revealed a transient up-regulation of the IL-4-induced CD23 mRNA by IFN-gamma within 2 h of IFN-gamma treatment in these tonsillar cells. The results suggest that the IL-4RE of the IL-4-inducible gene can accommodate both IL-4- and IFN-gamma-activated factors, such as STAT 6 and STAT 1, either in homodimeric or heterodimeric forms and the binding of these different proteins to the respective promoter may play a potential regulatory role in the IL-4-inducible gene expression.