Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

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Tribbles homolog 2 (Trib2) was identified as a downregulated transcript in leukemic cells undergoing growth arrest. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-transduced bone marrow cells exhibited a growth advantage ex vivo and readily established factor-dependent cell lines. In vivo, Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML). In mechanistic studies, we found that Trib2 associated with and inhibited C/EBPalpha. Furthermore, Trib2 expression was elevated in a subset of human AML patient samples. Together, our data identify Trib2 as an oncogene that induces AML through a mechanism involving inactivation of C/EBPalpha.

Gain-of-function experiments have demonstrated the potential of Notch signals to expand primitive hematopoietic progenitors, but whether Notch physiologically regulates hematopoietic stem cell (HSC) homeostasis in vivo is unclear. To answer this question, we evaluated the effect of global deficiencies of canonical Notch signaling in rigorous HSC assays. Hematopoietic progenitors expressing dominant-negative Mastermind-like1 (DNMAML), a potent inhibitor of Notch-mediated transcriptional activation, achieved stable long-term reconstitution of irradiated hosts and showed a normal frequency of progenitor fractions enriched for long-term HSCs. Similar results were observed with cells lacking CSL/RBPJ, a DNA-binding factor that is required for canonical Notch signaling. Notch-deprived progenitors provided normal long-term reconstitution after secondary competitive transplantation. Furthermore, Notch target genes were expressed at low levels in primitive hematopoietic progenitors. Taken together, these results rule out an essential physiological role for cell-autonomous canonical Notch signals in HSC maintenance.

Foxp3؉ T-regulatory cells (Tregs) are key to immune homeostasis such that their diminished numbers or function can cause autoimmunity and allograft rejection.

IntroductionNotch signaling plays a central role in multiple developmental processes. During blood and lymphoid development, Notch proteins influence several hematopoietic lineages. At the T-cell/B-cell branchpoint, Notch promotes T-cell at the expense of B-cell differentiation. 1,2 More recent data implicate Notch1 in the early generation of the hematopoietic stem cell (HSC) pool during embryogenesis 3 and Notch2 in marginal zone B-cell (MZB) development. [4][5][6] Notch family members also have been postulated to play a role in HSC self-renewal and homeostasis. 7,8 Other putative roles of Notch in the CD4/CD8 lineage choice, in ␣␤ versus ␥␦ T-cell differentiation and in the function of peripheral T cells are more controversial (reviewed in Radtke et al 9 and Maillard et al 10 ).Part of the lingering uncertainty over the role of Notch signals in hematopoiesis is related to the existence of multiple receptors and ligands with complex patterns of expression. Notch proteins are transmembrane receptors interacting with ligands of the Jagged and Delta-like families. Mammals have 4 different Notch receptors (Notch1-4) with a similar overall structure (reviewed in Radtke et al 9 and Maillard et al 10 ). Notch-mediated transcriptional activation involves proteolytic cleavage and physical translocation of the Notch intracellular domain (ICN) into the nucleus. Once in the nucleus, ICN binds to CSL (CBF1/RBP-Jk, Suppressor of Hairless, Lag-1), a transcription factor that mediates most of the wellcharacterized Notch functions. ICN binding has 2 consequences. First, it displaces co-repressors from CSL, resulting in derepression of promoters with CSL binding sites. 11 Secondly, ICN recruits Mastermind-like (MAML) proteins, 12 which appear to function as a scaffold for the formation of a large multiprotein transcriptional activation complex. 13 Three MAML family members (MAML1-3) have been identified in mammals. 14,15 MAMLs are nuclear proteins with short basic N-terminal sequences that bind the ankyrin repeat domain of ICN, and C-termini that recruit p300 and other transcriptional activators. In vitro experiments suggest that MAMLs are critical for Notch-mediated stimulation of CSL-dependent transcription 12,[14][15][16] and have shown that truncated MAML mutants consisting of only the N-terminal ICN-binding domain have potent dominant negative effects, presumably due to their inability to recruit other components of the Notch transcriptional activation complex. 16,17 The minimal region of MAML1 required for ICN binding, a basic region comprising amino acids 13 to 74, is sufficient to form a stable ternary complex with purified CSL and ICN (Nam et al 18 ) and when overexpressed in transfection assays, generates a profound block in Notch1-mediated transcriptional activation. 17 Furthermore, this minimal MAML1 13-74 region causes growth arrest and apoptosis of multiple T-ALL cell lines bearing constitutively active Notch1 alleles. 17 Although these data suggest that MAML proteins may be critical components of the Notch signali...

L-arginine causes insulin release from pancreatic B cells. Data from three model systems support the hypothesis that L-arginine-derived nitrogen oxides (NOs) mediate insulin release stimulated by L-arginine in the presence of D-glucose and by the hypoglycemic drug tolbutamide. The formation of NO in pancreatic B cells was detected both chemically and by the NO-induced accumulation of guanosine 3',5'-monophosphate. NG-substituted L-arginine analogs inhibited the release of both insulin and NO. Protein immunoblot and histochemical analysis with antiserum to type I NO synthase suggest that the formation of NO in pancreatic B cells is catalyzed by an NADPH- (reduced form of nicotinamide adenine dinucleotide phosphate), Ca2+/calmodulin-dependent type I NO synthase of about 150 kilodaltons.

BACKGROUND & AIMS Foxp3+ T regulatory cells (Tregs) help prevent autoimmunity, and increases in their numbers of functions could decrease the development of inflammatory bowel disease. Like other cells, Foxp3+ Tregs express histone/protein deacetylases (HDACs), which regulate chromatin remodeling and gene expression. We investigated whether disruption of a specific class IIa HDAC, HDAC9, activity in Tregs affects the pathogenesis of colitis in mice. METHODS We tested the effects of various HDAC inhibitors (HDACi) in models of colitis using wild-type mice. We also transferred Tregs and non-Treg cells from HDAC9−/− or wild-type mice to immunodeficient mice. HDAC9 contributions to the functions of Tregs were determined during development and progression of colitis. RESULTS Pan-HDACi, but not class I-specific HDACi, increased the functions of Foxp3+ Tregs, prevented colitis, and reduced established colitis in mice, indicating the role of class II HDACs in controlling Treg function. The abilities of pan-HDACi to prevent/reduce colitis were associated with increased numbers of Foxp3+ Tregs and their suppressive functions. Colitis was associated with increased local expression of HDAC9; HDAC9−/− mice resistant to development of colitis. HDAC9−/− Tregs expressed increased levels of the heat shock protein (HSP) 70, compared with controls. Immunoprecipitation experiments indicated an interaction between HSP70 and Foxp3. Inhibition of HSP70 reduced the suppressive functions of HDAC9−/− Tregs; Tregs that overexpressed HSP70 had increased suppressive functions. CONCLUSIONS Strategies to decrease HDAC9 expression or function in Tregs or to increase expression of HSP70 might be used to treat colitis and other autoimmune disorders.