Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization.

BACKGROUND: Ras homolog gene family H (RhoH) is a membrane-bound adaptor protein involved in proximal T-cell receptor signaling. Therefore RhoH plays critical roles in the differentiation of T cells; however, the function of RhoH in the effecter phase of the T-cell response has not been fully characterized. OBJECTIVE: We sought to explore the role of RhoH in inflammatory immune responses and investigated the involvement of RhoH in the pathogenesis of psoriasis. METHODS: We analyzed effector T-cell and systemic inflammation in wild-type and RhoH-null mice. RhoH expression in T cells in human PBMCs was quantified by using RT-PCR. RESULTS: RhoH deficiency in mice induced TH17 polarization during effector T-cell differentiation, thereby inducing psoriasis-like chronic dermatitis. Ubiquitin protein ligase E3 component N-recognin 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels decreased in RhoH-deficient T cells, resulting in increased protein levels and DNA binding activity of retinoic acid-related orphan receptor γt. The consequential increase in IL-17 and IL-22 production induced T cells to differentiate into TH17 cells. Furthermore, IL-22 binding protein/Fc chimeric protein reduced psoriatic inflammation in RhoH-deficient mice. Expression of RhoH in T cells was lower in patients with psoriasis with very severe symptoms. CONCLUSION: Our results indicate that RhoH inhibits TH17 differentiation and thereby plays a role in the pathogenesis of psoriasis. Additionally, IL-22 binding protein has therapeutic potential for the treatment of psoriasis.

A Histone Methyltransferase ESET Is Critical for T Cell Development.

ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET(-/-) thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET(-/-) thymocytes. Indeed, genetic depletion of FcγRIIB in ESET(-/-) thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET(-/-) mice. Therefore, impaired T cell development in ESET(-/-) mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.

MicroRNA-17~92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways.

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17~92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17~92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17~92. We experimentally identified miR-17~92 target genes by PAR-CLIP and validated select target genes in miR-17~92 transgenic mice. These analyses demonstrate that miR-17~92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17~92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17~92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

The thymic cortical epithelium determines the TCR repertoire of IL-17-producing γδT cells.

The thymus provides a specialized microenvironment in which distinct subsets of thymic epithelial cells (TECs) support T-cell development. Here, we describe the significance of cortical TECs (cTECs) in T-cell development, using a newly established mouse model of cTEC deficiency. The deficiency of mature cTECs caused a massive loss of thymic cellularity and impaired the development of αßT cells and invariant natural killer T cells. Unexpectedly, the differentiation of certain γδT-cell subpopulations-interleukin-17-producing Vγ4 and Vγ6 cells-was strongly dysregulated, resulting in the perturbation of γδT-mediated inflammatory responses in peripheral tissues. These findings show that cTECs contribute to the shaping of the TCR repertoire, not only of "conventional" αßT cells but also of inflammatory "innate" γδT cells.

Gasp, a Grb2-associating protein, is critical for positive selection of thymocytes.

T cells develop in the thymus through positive and negative selection, which are responsible for shaping the T cell receptor (TCR) repertoire. To elucidate the molecular mechanisms involved in selection remains an area of intense interest. Here, we identified and characterized a gene product Gasp (Grb2-associating protein, also called Themis) that is critically required for positive selection. Gasp is a cytosolic protein with no known functional motifs that is expressed only in T cells, especially immature CD4/CD8 double positive (DP) thymocytes. In the absence of Gasp, differentiation of both CD4 and CD8 single positive cells in the thymus was severely inhibited, whereas all other TCR-induced events such as beta-selection, negative selection, peripheral activation, and homeostatic proliferation were unaffected. We found that Gasp constitutively associates with Grb2 via its N-terminal Src homology 3 domain, suggesting that Gasp acts as a thymocyte-specific adaptor for Grb2 or regulates Ras signaling in DP thymocytes. Collectively, we have described a gene called Gasp that is critical for positive selection.

RhoH plays critical roles in Fc epsilon RI-dependent signal transduction in mast cells.

RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcepsilonRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcepsilonRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCgamma1, and PLCgamma2 were impaired, however phosphorylation of the gamma-subunit of FcepsilonRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcepsilonRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.

Rac1-mediated Bcl-2 induction is critical in antigen-induced CD4 single-positive differentiation of a CD4+CD8+ immature thymocyte line.

Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.

The atypical small GTPase RhoH : a novel role in T cell development.

Small GTPases (G-proteins) play important roles in various signal transduction pathways by working as molecular switches. Among them, some of these GTPases don't have functional features of typical GTPases, therefore they are called "atypical GTPases". Recently, these less known atypical Rho GTPases have received increased attention. This review will focus on the novel aspects of biological function of atypical Rho GTPases, especially a newly found function of RhoH on signal transduction in T cell development.

Regulation of B-cell development and tolerance by different members of the miR-17∼92 family microRNAs.

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.

Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, ß-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass ß-selection without TCRß gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

Differential function of Themis CABIT domains during T cell development.

Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection. Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved and controversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-ß in Themis) domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in Themis's function in vivo, we established a series of mutant Themis transgenic mice on a Themis(-/-) background. Deletion of the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2-association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus. The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not. Furthermore, expression of ΔCore1- but not ΔCore2-Themis conferred dominant negative-type inhibition on T cell development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive selection, and that each of the CABIT domains exerts distinct functions during positive selection.

Differential requirement for RhoH in development of TCRαß CD8αα IELs and other types of T cells.

RhoH is a new member of the atypical G proteins exclusively expressed in hematopoietic lineage cells. It has been shown to act as an adaptor for ZAP70, Syk, Lck and Csk kinases in signal transduction, and is required for positive selection of thymocytes as well as activation of peripheral T cells and mast cells. In the present study, we showed that RhoH is required not only for positive selection but also for negative selection of thymocytes. Regarding development of unconventional T cell subsets, development of NKT and regulatory T cells was also inhibited, whereas development of TCRαß CD8αα intestinal intraepithelial lymphocytes (IEL) was not affected by the absence of RhoH. TCR-dependent in vitro activation of TCRαß CD8αα IEL required RhoH, suggesting that overall development of IEL does not critically depend on TCR signaling but more on cytokine-dependent expansion and survival in the periphery. Our current results indicate differential requirements for RhoH in the development of TCRαß CD8αα IELs compared to other subsets of T cells including agonist selected T cells.

Rac GTPases are involved in development, survival and homeostasis of T cells.

Rac GTPases consist of Rac1, 2 and 3, and each of them have redundant and differential functions. Rac1 is the most ubiquitously and abundantly expressed of the three and has been shown to work as a "molecular switch" in various signal transduction pathways. Although Rac1 and Rac2 are both activated by TCR ligation, little is known about the function of Rac GTPases in the development and activation of T cells. In order to investigate the precise function of Rac GTPases in T cells in vivo, we established dominant negative Rac1 transgenic (dnRac1-Tg) mice controlled by the human CD2 promoter. Total numbers of thymocytes of dnRac1-Tg mice were significantly decreased because of impaired transition from the CD4CD8 double negative stage to the CD4CD8 double positive (DP) stage. Although positive selection of CD4 single positive (SP) was not altered, positive selection of CD8-SP was slightly increased. On the contrary, the number of mature CD4-SP and CD8-SP cells in the spleen, mesenteric lymph nodes and peripheral blood was severely decreased in dnRac1-Tg mice. Proliferation of splenic CD4-SP cells upon TCR stimulation in vitro was unaltered, however, homeostatic proliferation of dnRac1-Tg splenic CD4-SP cells in lymphopenic mice was severely reduced. Finally, we found increased spontaneous apoptosis of DP thymocytes and mature T cells in dnRac1-Tg mice, possibly because of reduced phosphorylation of Akt with or without TCR stimulation. Collectively, the current results indicate that Rac GTPases are important in survival of DP thymocytes and mature T cells in vivo by regulating Akt activation.

Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage: critical dimerization of inducible nitric-oxide synthase.

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.