Masamoto Kanno

IAN Family Critically Regulates Survival and Development of T Lymphocytes

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell–receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins.

Activation of Maf/AP-1 Repressor Bach2 by Oxidative Stress Promotes Apoptosis and Its Interaction with Promyelocytic Leukemia Nuclear Bodies

The oxidative stress response operates by inducing the expression of genes that counteract the stress. We show here that the oxidative stress-responsive transcription factor Bach2 is a generic inhibitor of gene expression directed by the 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element. The Bach2-enhanced green fluorescent protein bicistronic retrovirus was used to monitor the fate of Bach2-expressing cells at the single cell level. Bach2 exerted an inhibitory effect on NIH3T3 cell proliferation and caused massive apoptosis upon mild oxidative stress in both NIH3T3 and Raji B-lymphoid cells. Interestingly, Bach1, a highly homologous protein, could not induce cell death, demonstrating the specificity for the apoptosis induction. Although both oxidative stress and leptomycin B, an inhibitor of nuclear export, induce nuclear accumulation of Bach2, the leptomycin B-induced nuclear accumulation of Bach2 was not sufficient to elicit apoptosis. Upon oxidative stress, Bach2 formed nuclear foci that associated with promyelocytic leukemia nuclear bodies. Our results suggest that Bach2 constitutes a cell lineage-specific system that couples oxidative stress and cell death and that inhibition of 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element upon oxidative stress may be critical determinants for apoptosis.

Thymocyte proliferation induced by pre-T cell receptor signaling is maintained through polycomb gene product Bmi-1-mediated Cdkn2a repression.

Thymocytes undergo massive proliferation before T cell receptor (TCR) gene rearrangement, ensuring the diversification of the TCR repertoire. Because activated cells are more susceptible to damage, cell-death restraint as well as promotion of cell-cycle progression is considered important for adequate cell growth. Although the molecular mechanism of pre-TCR-induced proliferation has been examined, the mechanisms of protection against cell death during the proliferation phase remain unknown. Here we show that the survival of activated pre-T cells induced by pre-TCR signaling required the Polycomb group (PcG) gene product Bmi-1-mediated repression of Cdkn2A, and that p19Arf expression resulted in thymocyte cell death and inhibited the transition from CD4(-)CD8(-) (DN) to CD4(+)CD8(+) (DP) stage upstream of the transcriptional factor p53 pathway. The expression of Cdkn2A (the gene encoding p19Arf) in immature thymocytes was directly regulated by PcG complex containing Bmi-1 and M33 through the maintenance of local trimethylated histone H3K27. Our results indicate that this epigenetic regulation critically contributes to the survival of the activated pre-T cells, thereby supporting their proliferation during the DN-DP transition.

Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway

Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR β-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors isolated from mel-18−/− mice. In addition, mel-18−/− T precursors could not maintain the Hes-1 expression induced by Delta-like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors.

CELL DIFFERENTIATION AND p38MAPK CASCADE ARE INHIBITED IN HUMAN OSTEOBLASTS CULTURED IN A THREE-DIMENSIONAL CLINOSTAT

SummaryA three-dimensional (3D) clinostat is a device for multidirectional G force generation. By controlled rotation of two axes, a 3D clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10−3 G over time. We cultured a human osteoblast cell line in a 3D clinostat and examined the growth properties and differentiation of the cells, including morphology, histological detection of calcification, and mitogenactivated protein kinase (MAPK) cascades. In a normal 1G condition, alkaline phosphatase (AIPase) activity was detected on day 7 of culture, bone nodules were formed on day 12, and calcium deposits were seen on day 20. In the 3D clinostat, the cell looked larger and bulged. AIPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found in the 3D clinostat up to day 21. The expression levels of core-binding factor A1 (a transcription factor for bone formation) and osteocalcin (a bone matrix protein) increased in the control culture but decreased in culture in 3D clinostat. Phosphorylation of p38MARK (p38) was repressed in culture in 3D clinostat, whereas total p38 as well as total and phosphorylated forms of extracellular signal-regulated kinases and stress-activated protein kinase/jun N-terminal kinase were not changed in the 3D clinostat. When a p38 inhibitor, SB 203580, was added to the culture medium in a normal 1 G environment, AIPase activity and formation of bone nodules and calcium deposits were strongly inhibited. On the other hand, they were inhibited only partially by a MARK kinase inhibitor, U-0126. On the basis of these results, it is concluded that (1) osteoblast differentiation is inhibited in culture in a 3D clinostat and (2) this inhibition is mainly due to the suppression of p38 phophorylation.

The Role of the Basic Helix-Loop-Helix Transcription Factor Dec1 in the Regulatory T Cells

Naturally occurring regulatory T (Treg) cells play a central role in the maintenance of immune homeostasis and in restraining the development of spontaneous inflammatory responses. However, the underlying mechanisms of Treg homeostasis remain incompletely understood. Of particular note, the IL-2Rα (CD25) is crucial for the homeostasis of Treg cells and the prevention of lymphoproliferative autoimmune disease. In this paper, we report that the basic helix-loop-helix transcription factor Dec1 is involved in the homeostasis of Treg cells and plays a role in their survival or expansion after adoptive transfer to lymphopenic recipients. Hence, it is crucial for the suppression of effector T cell-mediated inflammatory responses. Enforced expression of Dec1 upregulates CD25 expression during thymocyte development and increases the number of Treg cells in the periphery. Dec1 binds the transcription factor Runx1 and colocalizes with Runx1 in Treg cells. Specifically, we demonstrate that in Treg cells the Dec1/Runx1 complex binds to regulatory elements present in the Il-2rα locus. Collectively, these data show how Dec1 mechanistically acts in Treg cells.

Epigenetic regulator polycomb group protein complexes control cell fate and cancer.

The chromatin‐associated Polycomb group (PcG) proteins were first identified in genetic screens for homeotic transformations in Drosophila melanogaster. Besides body patterning, members of the PcG are now known to regulate epigenetic cellular memory, stem cell self‐renewal, and cancer development. Here, we discuss the multifarious functions of the PcG family, isoforms of protein complexes, and its enzymatic activities, for example histone methylation, links to DNA methylation, its phosphorylation status, H2A mono‐ubiquitination, SUMOylation, and links to non‐coding RNA. We also discuss the function of cytosolic PcG complexes as a regulator of receptor‐induced actin polymerization and proliferation in a methylation‐dependent manner. We propose that the functional versatility of PcG protein complexes contributed significantly to the complexity of heritable gene repression mechanisms, signal transduction, and cell proliferation in cancer development. (Cancer Sci 2008; 99: 1077–1084)

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 gene expression by 22(R)-hydroxycholesterol requires de novo protein synthesis in activated macrophages

Liver X receptors (LXRs) play an important role in lipid metabolism. Recently, a role for these proteins was identified in suppressing the inflammatory response. However, it is not known whether the natural ligands of LXRs, e.g. 22(R)-hydroxycholesterol (22R-HC), can suppress the inflammatory response after the onset of inflammation. We demonstrate here that treatment of Lipopolysaccharide (LPS)-activated RAW264.7 macrophages with 22R-HC markedly suppressed nitric oxide (NO) production and inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression. Additionally, 22R-HC did not affect the DNA binding activity of NF-kappaB, AP-1 and C/EBP(s), important transcriptional factors for iNOS and COX-2 genes expression. Furthermore iNOS and COX-2 mRNA suppression by 22R-HC was diminished by cellular treatment with cycloheximide. These results suggest that 22R-HC suppresses the expression of iNOS and COX-2 genes through de novo protein synthesis of an unidentified protein in LPS-activated macrophages.

Dimerization of the Polycomb-group protein Mel-18 is regulated by PKC phosphorylation.

The Polycomb-group (Pc-G) gene products form complexes via protein-protein interactions and maintain the transcriptional repression of genes involved in embryogenesis, cell cycle, and tumorigenesis. Previously, we have shown that mouse Mel-18, a Pc-G protein, has tumor suppressor gene-like activity and negatively regulates transcription. Here, we show in vitro by pull-down assays and in vivo in transiently transfected COS-7 cells that Mel-18 forms homodimers. Deletion analysis revealed that the N-terminal RING-finger and alpha-helix domains are required for homodimer formation. In addition, we demonstrated that Mel-18 homo-dimerization is regulated by protein kinase C (PKC) and protein phosphatases, such that dephosphorylated Mel-18 is able to homo-dimerize. These results suggest that the stoichiometry and/or equilibrium of subunits of the class II Polycomb complex containing Mel-18 might be regulated by changes in phosphorylation status via the PKC signaling pathway.

The Endogenous Danger Signal Uric Acid Augments Contact Hypersensitivity Responses in Mice

Objective: The danger hypothesis proposes that the immune system responds not only to foreign antigens but also to damaged cells or tissues. Recently, uric acid crystals (monosodium urate, MSU) from necrotic cell lysates were identified as a danger signal for dendritic cells (DCs). Our aim was to determine whether MSU modulates immune responses in the skin. Method: We analyzed the effect of MSU on trinitrochlorobenzene-induced contact hypersensitivity responses using BALB/c mice administered potassium oxonate, an uricase inhibitor, to prevent MSU degradation. Ear swelling response after elicitation and activation profiles of DCs and T cells in draining lymph nodes after sensitization were assessed. Results: Intradermal administration of MSU augmented the ear swelling response in potassium oxonate-administered mice and enhanced expression of CD86 and CD40 molecules on DCs in the lymph nodes. Activation of DCs was followed by an increase in CD69+ and CD44+ T cells in CD4+ and/or CD8+ subsets in the lymph nodes 4 days after trinitrochlorobenzene sensitization. Conclusion: These observations demonstrate that MSU is an endogenous danger signal, which augments the contact hypersensitivity response in mice. MSU released from damaged skin may act as an endogenous adjuvant to augment immune response.