Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival

The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by ∼80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40–50% of CD4+Foxp3+ T cells vs. 7–8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival

HDAC7 Is a Repressor of Myeloid Genes Whose Downregulation Is Required for Transdifferentiation of Pre-B Cells into Macrophages

B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes

Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-κB-Dependent Transcription of Specific Genes after Genotoxic Stress

The NF-κB pathway is involved in immune and inflammation responses, proliferation, differentiation and cell death or survival. It is activated by many external stimuli including genotoxic stress. DNA double-strand breaks activate NF-κB in an ATM-dependent manner. In this manuscript, a direct interaction between p65(RelA) and the N-terminal extremity of ATM is reported. We also report that only one of the five potential ATM-(S/T)Q target sites present in p65, namely Ser547, is specifically phosphorylated by ATM in vitro. A comparative transcriptomic analysis performed in HEK-293 cells expressing either wild-type HA-p65 or a non-phosphorylatable mutant HA-p65S547A identified several differentially transcribed genes after an etoposide treatment (e.g. IL8, A20, SELE). The transcription of these genes is increased in cells expressing the mutant. Substitution of Ser547 to alanine does not affect p65 binding abilities on the κB site of the IL8 promoter but reduces p65 interaction with HDAC1. Cells expressing p65S547A have a higher level of histone H3 acetylated on Lys9 at the IL8 promoter, which is in agreement with the higher gene induction observed. These results indicate that ATM regulates a sub-set of NF-κB dependent genes after a genotoxic stress by direct phosphorylation of p65

Regulation of Glucose Homeostasis by KSR1 and MARK2

Protein scaffolds control the intensity and duration of signaling and dictate the specificity of signaling through MAP kinase pathways. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase cascade that regulates the intensity and duration of ERK activation. Relative to wild-type mice, ksr1-/- mice are modestly glucose intolerant, but show a normal response to exogenous insulin. However, ksr1-/- mice also demonstrate a three-fold increase in serum insulin levels in response to a glucose challenge, suggesting a role for KSR1 in insulin secretion. The kinase MARK2 is closely related to C-TAK1, a known regulator of KSR1. Mice lacking MARK2 have an increased rate of glucose disposal in response to exogenous insulin, increased glucose tolerance, and are resistant to diet-induced obesity. mark2-/-ksr1-/- (DKO) mice were compared to wild type, mark2-/-, and ksr1-/- mice for their ability to regulate glucose homeostasis. Here we show that disruption of KSR1 in mark2-/- mice reverses the increased sensitivity to exogenous insulin resulting from MARK2 deletion. DKO mice respond to exogenous insulin similarly to wild type and ksr1-/- mice. These data suggest a model whereby MARK2 negatively regulates insulin sensitivity in peripheral tissue through inhibition of KSR1. Consistent with this model, we found that MARK2 binds and phosphorylates KSR1 on Ser392. Phosphorylation of Ser392 is a critical regulator of KSR1 stability, subcellular location, and ERK activation. These data reveal an unexpected role for the molecular scaffold KSR1 in insulin-regulated glucose metabolism

Phosphorylation Regulates SIRT1 Function

BACKGROUND: SIR2 is an NAD(+)-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+)-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions

Effect Of Inhibitors Of Histone Deacetylase On The Induction Of Cell Differentiation In Murine And Human Erythroleukemia Cell Lines

Histone deacetylase (HDAC) inhibitors are a novel class of promising anti-cancer agents. Little information is available on the capacity of structurally different HDAC inhibitors to induce terminal cell differentiation in different cell types in relation to enzyme inhibition and subtype selectivity. Consequently, the aim of this study was to provide a comprehensive comparison of these effects. New biarylalanine inhibitors of HDAC were synthesized and compared to a series of standard inhibitors from different laboratories. Chromatographically purified rat liver and immunoprecipitated FLAG-tagged recombinant human HDACs were used as sources of HDAC activity. Enzyme inhibition was studied using a fluorescent substrate and its conversion was monitored by high-performance liquid chromatography. The ability to induce cell differentiation was compared in murine (Friend DS-19) and human (K562) erythroleukemic cell lines, and was quantified by benzidine staining. Inhibition of cell proliferation was evaluated by cell counting. All HDAC inhibitors were identified as potent inhibitors of erythroleukemic cell proliferation. However, we observed a complex pattern of differentiation induction: structurally similar inhibitors resulted in disparate activity profiles, whereas similar profiles were detected within distinct structural classes. Among the newly synthesized biarylalanine compounds, a 3'-methoxy derivative was identified as a very effective inducer of terminal cell differentiation. We conclude that investigation of subtype selectivity of selected HDAC inhibitors does not provide a clear link between selectivity and the observed cellular activity profile. The predictive value of in vitro HDAC inhibition assays for identifying anti-proliferative compounds has been emphasized