Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by proteintyrosine phosphatases (PTPs). IntroductionAcute myeloid leukemia (AML) is the most frequent leukemia in adults with improving but still limited treatment possibilities, notably in elderly patients. 1,2 It arises by malignant transformation of myeloid progenitor cells. Among the contributing genetic lesions, mutations in the class III receptor tyrosine kinase (RTK) FMS-like tyrosine kinase 3 (FLT3) occur in approximately 30% of patients. 3 The prevalent type of FLT3 mutations are internal tandem duplications (ITD) of amino acid stretches in the juxtamembrane domain or in the tyrosine kinase domain, 4,5 which confer cytokineindependent proliferation and resistance to apoptosis, and causally contribute to AML in combination with additional genetic lesions. 1 Compared with the ligand-activated wild-type (WT) FLT3, FLT3 ITD mutants exhibit not only elevated but also altered signaling quality, with very pronounced activation of signal transducer and activator of transcription (STAT)5 as one characteristic feature. 6,7 FLT3 ITD also causes the production of high levels of reactive oxygen species (ROS). 8,9 Signal transduction of RTKs is regulated by protein-tyrosine phosphatases (PTPs). PTPs prevent ligand-independent RTK activation, and contribute to modulation and termination of ligand-induced signaling. 10 The activity of PTPs is regulated at several different levels. 11 One regulatory principle is the reversible oxidation of the PTP active-site cysteine, which leads to reversible inactivation. 12,13 Temporary inactivation of negatively regulating PTPs by this mechanism is believed to be important for efficient RTK signal propagation in the cell. 14 A major ROS causing cellular PTP oxidation is hydrogen peroxide (H 2 O 2 ), which can be generated by a dismutase reaction from superoxide anions, the reaction products of NADPH-oxidases. Activation of the NADPH oxidase isoform 1 (NOX1) occurs downstream of RTK activation, and involves activation and membrane translocation of the small guanosine triphosphate (GTP)ase Rac1. 15 ROS generation in the cell is counteracted by efficient ROS decomposing systems. 16 These include peroxiredoxins (Prx), which have a very low K m for H 2 O 2 and can eliminate it even at low concentrations. 17 Relatively little is known about PTPs regulating FLT3 signal transduction. We have previously shown that the nontransmembrane PTPs PTP1B and SHP-1 can potently dephosphorylate FLT3 on overexpression. Further, PTP1B appears important for suppressing signaling of newly synthesized FLT3. 7,18 SHP-2 acts as a positive regulator, because it is important for Erk activation and proliferation induced by ligand-activated WT FLT3. However, it is dispensable for FLT3 ITD-mediated transformation. 19 We previously performed a shRNA-based screen to identify PTPs regulating WT FLT3. The initial screen assessed the effects of shRNAs for 20 PTPs on FL-induced Erk1/2 activation in WT FLT3-expressing 32D cells. Among several potentia...

Phosphorylation of wild type FLT3 and AML-associated mutant FLT3 was recently analyzed using site-specific phosphotyrosine antibodies (15). Interestingly, the phosphorylation pattern of the different FLT3 variants showed quantitative and also qualitative differences. Although FLT3-ITD or mutations in the kinase domain resulted in ligand-independent FLT3 autophosphorylation and signaling activity, the wild type receptor is only autophosphorylated in response to stimulation with its cytokine FL.Signaling of receptor tyrosine kinases is modulated by protein-tyrosine phosphatases (PTP) (16), and aberrations in PTP function play a role in carcinogenesis (17). Some PTP, notably SHP-2, have been found to positively influence growth-stimulatory signaling pathways, and mutations leading to gain-offunction of these PTP can potentially be oncogenic. It has been demonstrated that SHP-2 directly interacts with FLT3 in a phosphorylation-dependent manner via phosphotyrosine 599. Table S1. 1 To whom correspondence should be addressed. Tel.: 49-3641-9395634; Fax:49-3641-9395602; E-mail: joerg.mueller2@med.uni-jena.de.2 The abbreviations used are: AML, acute myeloid leukemia; PTP, proteintyrosine phosphatase; FL, FLT3 ligand; PLC␥, phospholipase C␥; ITD, internal tandem duplication; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Introduction: Cells of the innate immune system particularly monocytes and macrophages have been recognized as pivotal players both during the initial insult as well as the chronic phase of atherosclerosis. It has recently been shown that oxidized low-density lipoprotein (oxLDL) induces a long-term pro-inflammatory response in monocytes due to epigenetic and metabolic reprogramming, an emerging new concept called trained innate immunity. Changes in the cellular redox state are crucial events in the regulation of many physiologic functions in macrophages including transcription, differentiation and inflammatory response. Here we have analyzed the role of reactive oxygen species (ROS) in regulating this proinflammatory monocyte priming in response to oxLDL-treatment.Methods and Results: Human monocytes were isolated and incubated with oxLDL for 24 h. After 5 days of resting, oxLDL treated cells produced significantly more inflammatory cytokines upon restimulation with the TLR2-agonist Pam3cys. Furthermore, oxLDL incubation induced persistent mTOR activation, ROS formation, HIF1α accumulation and HIF1α target gene expression, while pharmacologic mTOR inhibition or siRNA mediated inhibition of the mTORC1 subunit Raptor prevented ROS formation and proinflammatory priming. mTOR dependent ROS formation was associated with increased expression of NAPDH oxidases and necessary for the emergence of the primed phenotype as antioxidant treatment blocked oxLDL priming. Inhibition of cytosolic ROS formation could also block mTOR activation and HIF1α accumulation suggesting a positive feedback loop between mTOR and cytosolic ROS. Although mitochondrial ROS scavenging did not block HIF1α-accumulation at an early time point (24 h), it was persistently reduced on day 6. Therefore, mitochondrial ROS formation appears to occur initially downstream of the mTOR-cytoROS-HIF1α feedback loop but seems to be a crucial factor that controls the long-term activation of the mTOR-HIF1α-axis.Conclusion: In summary, our data demonstrate that mTOR dependent ROS production controls the oxLDL-induced trained innate immunity phenotype in human monocyte derived macrophages. Pharmacologic modulation of these pathways might provide a potential approach to modulate inflammation, associated with aberrant monocyte activation, during atherosclerosis development.

Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H2O2, as well as biotic- and abiotic-induced redox signals. RRTF1 is highly conserved in angiosperms, but its physiological role remains elusive. Here we show that inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Transgenic lines overexpressing RRTF1 are impaired in root and shoot development, light sensitive, and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica, which reduces ROS accumulation locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors that scavenge ROS. More than 800 genes were detected in mature leaves and seedlings of transgenic lines overexpressing RRTF1; ∼ 40% of them have stress-, redox-, ROS-regulated-, ROS-scavenging-, defense-, cell death- and senescence-related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box-like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli and H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains a GCC-box-like sequence in its promoter, but transgenic lines overexpressing RAP2.6 do not accumulate higher ROS levels. RRTF1 also stimulates systemic ROS accumulation in distal non-stressed leaves. We conclude that the elevated levels of the highly conserved RRTF1 induce ROS accumulation in response to ROS and ROS-producing abiotic and biotic stress signals.