Shunsuke Kon

The Runx3 Transcription Factor Augments Th1 and Down-Modulates Th2 Phenotypes by Interacting with and Attenuating GATA3

Recently, it was reported that the expression of Runt-related transcription factor 3 (Runx3) is up-regulated in CD4+ helper T cells during Th1 cell differentiation, and that Runx3 functions in a positive feed-forward manner with the T-box family transcription factor, T-bet, which is a master regulator of Th1 cell differentiation. The relative expression levels of IFN-γ and IL-4 are also regulated by the Th2-associated transcription factor, GATA3. Here, we demonstrate that Runx3 was induced in Th2 as well as Th1 cells and that Runx3 interacted with GATA3 and attenuated GATA3 transcriptional activity. Ectopic expression of Runx3 in vitro in cultured cells or transgenic expression of Runx3 in mice accelerated CD4+ cells to a Th1-biased population or down-modulated Th2 responses, in part by neutralizing GATA3. Our results suggest that the balance of Runx3 and GATA3 is one factor that influences the manifestation of CD4+ cells as the Th1 or Th2 phenotypes.

Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1

E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition.

EPLIN is a crucial regulator for extrusion of RasV12- transformed cells

ABSTRACT At the initial stage of carcinogenesis, a mutation occurs in a single cell within a normal epithelial layer. We have previously shown that RasV12-transformed cells are apically extruded from the epithelium when surrounded by normal cells. However, the molecular mechanisms underlying this phenomenon remain elusive. Here, we demonstrate that Cav-1-containing microdomains and EPLIN (also known as LIMA1) are accumulated in RasV12-transformed cells that are surrounded by normal cells. We also show that knockdown of Cav-1 or EPLIN suppresses apical extrusion of RasV12-transformed cells, suggesting their positive role in the elimination of transformed cells from epithelia. EPLIN functions upstream of Cav-1 and affects its enrichment in RasV12-transformed cells that are surrounded by normal cells. Furthermore, EPLIN regulates non-cell-autonomous activation of myosin-II and protein kinase A (PKA) in RasV12-transformed cells. In addition, EPLIN substantially affects the accumulation of filamin A, a vital player in epithelial defense against cancer (EDAC), in the neighboring normal cells, and vice versa. These results indicate that EPLIN is a crucial regulator of the interaction between normal and transformed epithelial cells.

Runx1 Deficiency in CD4+ T Cells Causes Fatal Autoimmune Inflammatory Lung Disease Due to Spontaneous Hyperactivation of Cells

The Runx1 transcription factor is abundantly expressed in naive T cells but rapidly downregulated in activated T cells, suggesting that it plays an important role in a naive stage. In the current study, Runx1−/−Bcl2tg mice harboring Runx1-deleted CD4+ T cells developed a fatal autoimmune lung disease. CD4+ T cells from these mice were spontaneously activated, preferentially homed to the lung, and expressed various cytokines, including IL-17 and IL-21. Among these, the deregulation of IL-21 transcription was likely to be associated with Runx binding sites located in an IL-21 intron. IL-17 produced in Runx1-deleted cells mobilized innate immune responses, such as those promoted by neutrophils and monocytes, whereas IL-21 triggered humoral responses, such as plasma cells. Thus, at an initial stage, peribronchovascular regions in the lung were infiltrated by CD4+ lymphocytes, whereas at a terminal stage, interstitial regions were massively occupied by immune cells, and alveolar spaces were filled with granular exudates that resembled pulmonary alveolar proteinosis in humans. Mice suffered from respiratory failure, as well as systemic inflammatory responses. Our data indicate that Runx1 plays an essential role in repressing the transcription of cytokine genes in naive CD4+ T cells and, thereby, maintains cell quiescence.

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML.

KSRP/FUBP2 Is a Binding Protein of GO-Y086, a Cytotoxic Curcumin Analogue

Bis(arylmethylidene)acetone derivatives are an important class of curcumin analogues that exhibit various biological and pharmacological activities. We herein report that GO-Y086, a biotinylated bis(arylmethylidene)acetone, inhibits cancer cell growth. We also show that GO-Y086 specifically interacts with the nuclear protein KSRP/FUBP2 by covalent modification. GO-Y086 markedly suppresses the expression of the c-Myc protein, which plays an important role in cellular proliferation and whose expression is regulated by KSRP/FUBP2.

Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: implications in cancer cell biology.

The endocytosis of cell membrane proteins is initiated by the binding of activated Arf6, a member of Ras‐related GTPases, to the PM. A GAP specific for Arf6 triggers the budding of endocytotic vesicles from the PM by inactivating GTP‐bound Arf6. We recently identified the SMAP gene that encodes an ArfGAP and is involved in the endocytosis of TfnR and possibly E‐cadherin. In this review, we summarize the process of intracellular membrane trafficking, highlighting the roles played by the SMAP gene. Progression of cancer to malignancy occurs in parallel with the disappearance of E‐cadherin, a central component of the adherens junction in epithelial cells. Therefore, elucidation of the molecular mechanism of E‐cadherin endocytosis should be one of the key elements in tumor cell biology. (Cancer Sci 2006; 97: 801–806)

The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis

SMAP2 is an Arf GAP and modulates clathrin-coated vesicle formation. SMAP2-deficient male mice exhibited globozoospermia due to acrosome deformation. In SMAP2(−/−) spermatids, budding of proacrosomal vesicles from the TGN was distorted and clathrin traffic–related molecules such as CALM and syntaxin2 were mislocated.

A role of the sphingosine-1-phosphate (S1P)- S1P receptor 2 pathway in Epithelial Defense Against Cancer (EDAC)

Normal epithelial cells have an ability to sense and actively eliminate neighboring transformed cells, a process called epithelial defense against cancer (EDAC). Exogenous S1P plays a crucial role in EDAC; the S1P–S1PR2 pathway regulates Rho–Rho kinase–filamin in the surrounding normal cells, promoting apical extrusion of RasV12-transformed cells from epithelia.

T‐cell receptor signaling induces proximal Runx1 transactivation via a calcineurin–NFAT pathway

Runx1 transcription factor is a key player in the development and function of T cells. Runx1 transcripts consist of two closely related isoforms (proximal and distal Runx1) whose expressions are regulated by different promoters. Which Runx1 isoform is expressed appears to be tightly regulated. The regulatory mechanism for differential transcription is, however, not fully understood. In this study, we investigated the regulation of the proximal Runx1 promoter in T cells. We showed that proximal Runx1 was expressed at a low level in naïve T cells from C57BL/6 mice, but its expression was remarkably induced upon T‐cell activation. In the promoter of proximal Runx1, a highly conserved region was identified which spans from −412 to the transcription start site and harbors a NFAT binding site. In a luciferase reporter assay, this region was found to be responsive to T‐cell activation through Lck and calcineurin pathways. Mutagenesis studies and chromatin immunoprecipitation assay indicated that the NFAT site was essential for NFAT binding and transactivation of the proximal Runx1 promoter. Furthermore, TCR signaling‐induced expression of proximal Runx1 was blocked by treatment of cells with cyclosporin A. Together, these results demonstrate that the calcineurin–NFAT pathway regulates proximal Runx1 transcription upon TCR stimulation.