TSCOT+ thymic epithelial cell-mediated sensitive CD4 tolerance by direct presentation.

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.

Tumor suppressor VDUP1 increases p27(kip1) stability by inhibiting JAB1.

Vitamin D3 up-regulated protein 1 (VDUP1) is a stress-response gene that is up-regulated by 1,25(OH)2D3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1-/- fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27(kip1), a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27(kip1) and to decrease the stability of p27(kip1). VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27(kip1) stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27(kip1) from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27(kip1) stability via regulating JAB1.

VDUP1 is required for the development of natural killer cells.

Vitamin D3 upregulated protein 1 (VDUP1) is a stress-response gene that is upregulated by 1,25(OH)2D3 in tumor cells. The in vivo roles of VDUP1 were investigated by producing mice lacking VDUP1 (VDUP1-/- mice). VDUP1-/- mice showed minimal changes in the development of T and B cells, but there was a profound reduction in the numbers of natural killer (NK) cells. As well, these mice showed decreased NK activity. In the VDUP1-/- mice, the expression of CD122 was reduced, demonstrating that VDUP1 is required for CD122 expression and NK maturation. In addition, severe lymphoid hyperplasia in the small intestine was observed in VDUP1-/- mice. Taken together, these results suggest that VDUP1 is a critical factor for the development and function of NK cells in vivo.

Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway.

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-alpha-induced activation of NF-kappaB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-alpha-induced activation of NF-kappaB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-alpha for NF-kappaB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-kappaB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-kappaB activation triggered by TNF-alpha and caspase-8. In addition, expression of an NF-kappaB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-alpha and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-kappaB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-alpha-induced activation of NF-kappaB via FLASH.

Aberrant signaling of TGF-beta1 by the mutant Smad4 in gastric cancer cells.

TGF-beta1 has been known to suppress the growth of gastric cancer cells. Interestingly, TGF-beta1 treatment increased the proliferation of human gastric cancer cell line, SNU-216 cells, while it reduced the proliferation of other tumor cells including SNU-620 cells. TGF-beta1-mediated down-regulation of c-Myc and induction of p21CIP1 were observed in SNU-620, but there was no change in SNU-216 in response to TGF-beta1. Similarly, TGF-beta1 receptors were upregulated by TGF-beta1 treatment in SNU-620, but they were not responded in SNU-216. By a single strand conformation polymorphism analysis, a repeated insertion of 37 nucleotides in the exon 8 of Smad4, resulting in premature termination at codon 362, was found in SNU-216. Furthermore, this truncated Smad4 functioned as a dominant negative form in TGF-beta1-mediated reporter activity and TGF-beta1 receptor expression. However, the proliferation of tumor cells was not affected by Smad4 mutation, but it was modulated by PD98059. Taken together, a mutation in Smad4 in addition to mitogen-activated protein kinase altered the TGF-beta1-mediated signaling, which is one of key events of gastric tumorigenesis.

VDUP1 upregulated by TGF-beta1 and 1,25-dihydorxyvitamin D3 inhibits tumor cell growth by blocking cell-cycle progression.

Vitamin D(3) upregulated protein 1 (VDUP1) is a 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) upregulated protein, and it is induced by various stresses. In human tumor tissues, VDUP1 expression was downregulated. Upon stimulation by growth-inhibitory signals such as TGF-beta1 and 1,25(OH)(2)D(3), its expression was rapidly upregulated as the cell growth was retarded. The transfection of VDUP1 in tumor cells reduced cell growth. The VDUP1 expression was also increased when the cell-cycle progression was arrested. Transfection of VDUP1 induced cell-cycle arrest at the G0/G1 phase, indicating that VDUP1 possesses a tumor-suppressive activity. In addition, it was found that VDUP1 interacted with promyelocytic leukemia zinc-finger, Fanconi anemia zinc-finger, and histone deacetylase 1, which are known to be transcriptional corepressors. VDUP1 itself suppressed IL-3 receptor and cyclin A2 promoter activity. Taken together, these results suggest that VDUP1 is a novel antitumor gene which forms a transcriptional repressor complex.