Masashi Fukasawa

Regulation of Nuclear Import/Export of Carbohydrate Response Element-binding Protein (ChREBP) INTERACTION OF AN α-HELIX OF ChREBP WITH THE 14-3-3 PROTEINS AND REGULATION BY PHOSPHORYLATION

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.

Age-Associated Augmentation of the Synthetic Ligand- Mediated Function of Mouse NK1.1 Ag+ T Cells: Their Cytokine Production and Hepatotoxicity In Vivo and In Vitro

We recently reported that the direct antitumor effectors in the liver induced by α-galactosylceramide (α-GalCer) are NK cells that are activated by the IFN-γ produced from NK1.1 Ag+ T cells (NKT cells) specifically stimulated with α-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the α-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-γ and IL-4 concentrations as well as alanine aminotransferase levels after the α-GalCer injection increased in an age-dependent manner. An α-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of α-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-γ and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1+ cells. Furthermore, liver mononuclear cells from old mice stimulated with α-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most α-GalCer-injected old mice, but no young mice, died, while anti-IFN-γ Ab pretreatment completely inhibited mouse mortality. However, α-GalCer-induced hepatic injury did not improve at all by anti-IFN-γ Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-γ is responsible for α-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-γ.

Functional and Vβ repertoire characterization of human CD8+ T‐cell subsets with natural killer cell markers, CD56+ CD57− T cells, CD56+ CD57+ T cells and CD56− CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK‐type T cell); CD56 single positive (CD56)‐T cells, CD56/CD57 double positive (DP)‐T cells and CD57 single positive (CD57)‐T cells in the peripheral blood. All NK‐type T‐cell populations expressed CD122 and intermediate levels of T‐cell receptor (TCR; regular CD8+ T cells are CD122− and express high levels of TCR). The number of both DP‐T cells and CD57‐T cells, but not CD56‐T cells, gradually increased with age. All NK‐type T‐cell populations produced larger amounts of interferon‐γ than did regular CD8+ T cells after stimulation with interleukin (IL)‐2, IL‐12 and IL‐15. However, CD56‐T cells and CD57‐T cells but not DP‐T cells showed a potent antitumour cytotoxity to NK‐sensitive K562 cells, whereas only CD56‐T cells showed a potent cytotoxity to NK‐resistant Raji cells. Furthermore, although NK‐type T cells produced large amounts of soluble Fas‐ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VβT cells were found in each NK‐type T‐cell population. The non‐variant CDR3 region(s) for the TCRβ chain(s) showed CD57‐T cells and CD56‐T cells to be derived from distinct origins, while the DP‐T cell population consisted of a mixture of the clones seen in both CD56‐T cells and CD57‐T cells. Our results suggest that CD57‐T cells and CD56‐T cells are functionally and ontogenically different populations while DP‐T cells appear to originate from both CD56‐T cells and CD57‐T cells.

Enhancement of the synthetic ligand‐mediated function of liver NK1.1Ag+ T cells in mice by interleukin‐12 pretreatment

We previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin‐12 (IL‐12) act as anti‐tumour/anti‐metastatic effectors. However, IL‐12 reportedly induces a rapid disappearance of liver NKT cells by activation‐induced apoptosis. In the present study, however, we show that injection of IL‐12 into mice merely down‐regulates the NK1.1 expression of liver NKT cells and Vβ8+ intermediate T‐cell receptor cells and CD1d/α‐galactosylceramide (α‐GalCer)‐tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL‐12‐pretreated (24 hr before) mice were injected with α‐GalCer, not only serum interferon‐γ but also serum IL‐4 concentrations increased several‐fold in comparison to the control α‐GalCer‐injected mice. However, IL‐12 pretreatment markedly up‐regulated serum ALT levels and Fas‐ligand expression on NKT cells after α‐GalCer injection in middle‐aged mice only. Consistently, the liver mononuclear cells (MNC) from IL‐12‐pretreated mice stimulated with α‐GalCer in vitro produced much greater amounts of interferon‐γ and IL‐4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate‐buffered saline. Liver MNC from middle‐aged mice, but not from young mice pretreated with IL‐12, also showed increased cytotoxicity following in vitroα‐GalCer stimulation against cultured hepatocytes. Furthermore, IL‐12 treatment of middle‐aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vβ8+ T cells, and in vitro experiments also revealed that IL‐12 pretreatment of liver MNC from middle‐aged mice enhanced their tumour necrosis factor‐α production after α‐GalCer stimulation. Synthetic ligand‐mediated functions of NKT cells, including IL‐4 production, are thus enhanced by IL‐12 pretreatment.

IL-18 time-dependently modulates Th1/Th2 cytokine production by ligand-activated NKT cells.

While IL‐18 synergizes with IL‐12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL‐18 on the Th1/Th2 cytokine response. The injection of α‐galactosylceramide (α‐GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN‐γ and IL‐4. When the mice were treated 2 h before α‐GalCer challenge with IL‐18, IFN‐γ production but not IL‐4 production was remarkably up‐regulated. In contrast, pretreatment with IL‐18 6 h before the challenge enhanced IL‐4 production. However, this IL‐18‐enhanced IL‐4 production was not elicited in mice injected with anti‐CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL‐18 either 2 h or 6 h before challenge were stimulated with α‐GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up‐regulated in the liver MNC from mice pretreated 6 h before with IL‐18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up‐regulated the phosphorylation of STAT3 and suppressed in vitro IL‐4 production by IL‐18‐primed liver MNC stimulated with α‐GalCer, but it did not affect IFN‐γ production. These results suggest that IL‐18 time‐dependently modulates Th1/Th2 cytokine production in ligand‐activated NKT cells by regulating/inducing SOCS3 expression.

Analysis of the susceptibility of CD57+ T cells to CD3-mediated apoptosis

After stimulation with anti‐CD3 antibody in vitro, CD57+ T cells showed a greater susceptibility to apoptosis than CD57–αβT cell receptor (TCR)+ T cells (regular αβ T cells). The apoptotic fraction of CD57+ T cells showed an increased production of active caspase‐3. An increase in both Fas expression and Fas‐ligand (FasL) production was also observed in CD57+ T cells, whereas the expression of survivin was suppressed in CD57+ T cells compared to that of regular αβ T cells. CD57+ T cells display a biased expansion of a few Vβ T cell fractions in individuals, but such Vβ T cells were not specifically susceptible to CD3‐mediated apoptosis. The TCR expression level of CD57+ T cells was much lower than that of regular T cells and anti‐TCR antibody stimulation induced a smaller apoptotic proportion of CD57+ T cells than did anti‐CD3 antibody. Although the CD3ɛ expression levels were similar in both T cell subsets, the CD3ζ level of CD57+ T cells was significantly higher than that of regular T cells. These results suggest that several apoptotic and anti‐apoptotic molecules are involved in the CD3‐induced apoptosis of CD57+ T cells and raise the possibility that the imbalance in expression of the CD3ɛ and CD3ζ chains may also contribute to the susceptibility of CD57+ T cells to undergo apoptosis.

Interplay between ChREBP and SREBP-1c coordinates postprandial glycolysis and lipogenesis in livers of mice

Lipogenesis in liver is highest in the postprandial state; insulin activates SREBP-1c, which transcriptionally activates genes involved in FA synthesis, whereas glucose activates carbohydrate-responsive element-binding protein (ChREBP), which activates both glycolysis and FA synthesis. Whether SREBP-1c and ChREBP act independently of one another is unknown. Here, we characterized mice with liver-specific deletion of ChREBP (L-Chrebp−/− mice). Hepatic ChREBP deficiency resulted in reduced mRNA levels of glycolytic and lipogenic enzymes, particularly in response to sucrose refeeding following fasting, a dietary regimen that elicits maximal lipogenesis. mRNA and protein levels of SREBP-1c, a master transcriptional regulator of lipogenesis, were also reduced in L-Chrebp−/− livers. Adeno-associated virus-mediated restoration of nuclear SREBP-1c in L-Chrebp−/− mice normalized expression of a subset of lipogenic genes, while not affecting glycolytic genes. Conversely, ChREBP overexpression alone failed to support expression of lipogenic genes in the livers of mice lacking active SREBPs as a result of Scap deficiency. Together, these data show that SREBP-1c and ChREBP are both required for coordinated induction of glycolytic and lipogenic mRNAs. Whereas SREBP-1c mediates insulin’s induction of lipogenic genes, ChREBP mediates glucose’s induction of both glycolytic and lipogenic genes. These overlapping, but distinct, actions ensure that the liver synthesizes FAs only when insulin and carbohydrates are both present.