Akemi Hoshino

Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC–UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.

Regulation of Cell Proliferation by Interleukin-3-induced Nuclear Translocation of Pyruvate Kinase

Extracellular signaling molecules bound to cell surface receptors can regulate nuclear function with consequences for cell proliferation, differentiation, and function. To regulate nuclear function, signals must be transduced across the nuclear envelope to propagate the signal from the cytoplasm to the nucleus. Therefore, many signaling responses induce the nuclear translocation of transcription factors, kinases, and others. By using inducible translocation trap, a reporter gene-based system to detect inducible nuclear translocation, we found that the M2 isoform of pyruvate kinase, a key enzyme in glycolysis, translocates into the nucleus by interleukin-3, but not by epidermal growth factor, stimulation. The C domain of the M2 isoform of pyruvate kinase was sufficient for interleukin-3-induced nuclear translocation. Interleukin-3-induced nuclear translocation of the M2 isoform of pyruvate kinase was dependent on the activation of Jak2. Overexpression of the M2 isoform of pyruvate kinase protein fused with a nuclear localization signal enhanced cell proliferation in the absence of interleukin-3, suggesting that the nuclear pyruvate kinase plays an important role in cell proliferation.

Insertional chromatin immunoprecipitation: A method for isolating specific genomic regions

We established a novel method, insertional chromatin immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting chromatin structure of genomic region of interest.

Redundant promoter elements mediate IL-3-induced expression of a novel cytokine-inducible gene, cyclon

Cytokines control cell differentiation, proliferation, and function by regulating gene expression program. Physiological roles and induction mechanisms of cytokine‐inducible genes are not fully understood. Here, we identified a novel immediate‐early cytokine‐responsive gene, cyclon (cytokine‐induced protein with coiled‐coil domain), which is induced in a hematopoietic cell line by interleukin 3 (IL‐3). cyclon gene encodes a phosphorylated nuclear protein consisting of repetitive sequences in the amino‐terminus and a coiled‐coil domain in the carboxyl‐terminus. A novel transient reporter assay revealed that mouse cyclon promoter contains redundant elements for IL‐3‐induced gene expression.

Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon

Activation-induced cell death (AICD) plays an essential role in the contraction of activated T cells after eradication of pathogen. Fas (APO-1/CD95) is one of the key cell surface proteins that mediate AICD in CD4(+) and CD8(+) T cells. Despite its prime importance in cell death, regulation of Fas expression in T cells is poorly understood. Here we show that Cyclon, a newly identified cytokine-inducible protein, is induced in T cells on T-cell receptor ligation and important for immune homeostasis. Transgenic expression of Cyclon ameliorated autoimmune phenotype in mice lacking subunits of IL-2R. Transgenic expression of Cyclon markedly enhanced AICD through increased expression of Fas whose expression is essential for Cyclon action. Finally, we demonstrated that activated but not resting CD4(+) T cells with targeted deletion of a Cyclon allele show reduced AICD and expression of Fas, indicating a critical role of Cyclon in Fas expression in activated T cells. We think that our data provide insight into expression regulation of Fas in T cells.

Induction of synaptic depression by high-frequency stimulation in area CA1 of the rat hippocampus: Modeling and experimental studies

It is generally accepted that low-frequency stimulation (LFS) induces long-term depression (LTD), while high-frequency stimulation (HFS) induces long-term potentiation (LTP). We performed modeling and experimental studies to see whether this commonly accepted view is the case for a wide range of stimulation frequencies, and found the induction of LTD by high-frequency stimulation of 300Hz (HFS-LTD) both in simulation and experiments. HFS-LTD was occluded by standard LFS-LTD induced by 1Hz-15min stimulation, and blocked by D-AP5, W-7 or FK-506, but neither by Ni^+ nor nimodipine. The analysis of the model suggested that the crossing of activation curve of CaMKII twice with that of CaN as the increase in [Ca^2^+]i was a possible mechanism for the induction of HFS-LTD in our study.

Nuclear translocation of 2 amino 3 ketobutyrate coenzyme A ligase by cold and osmotic stress

Abstract Cells are continuously exposed to environmental stresses and respond to them to maintain cellular homeostasis. Failure to respond to these stresses may cause pathological states such as renal failure, complications of diabetes, and autoimmune diseases. Signal transduction induced by osmotic and cold stresses is not fully understood. In addition, mechanisms of these stress responses are yet to be elucidated. Activation of many signaling pathways induces translocation of proteins into the nucleus to transduce signals and regulate nuclear functions. By using inducible translocation trap (ITT), a reporter gene–based screening technique, nuclear translocation of 2-amino-3-ketobutyrate coenzyme A ligase (KBL) was detected in response to cold and osmotic stresses. Rapid nuclear translocation of KBL was confirmed by biochemical analysis and fluorescent microscopy. A large region of KBL was required for stress-induced nuclear translocation. The KBL reporter system will be a useful tool for the investigation of cold and osmotic stress responses.

Lack of nuclear translocation of cytoplasmic domains of IL-2/IL-15 receptor subunits

Some sensors of extracellular signaling molecules such as Notch and sterol response element binding protein (SREBP) receive ligand-induced intra-membrane proteolysis followed by nuclear translocation of their cytoplasmic domains to regulate gene expression programs in the nucleus. It has not been extensively examined whether ligand-induced intra-membrane proteolysis of type I cytokine receptors and nuclear translocation of cytoplasmic domains occur. Here, by using a sensitive reporter system, we examined this possibility for the interleukin-2 (IL-2) receptor (IL-2R) beta-chain (IL-2R beta) and the IL-15 receptor (IL-15R) alpha-chain (IL-15R alpha). Flowcytometric analysis revealed that ligand stimulation does not induce nuclear translocation of their cytoplasmic domains. In addition, overexpression of the cytoplasmic domain of the common cytokine receptor gamma-chain (gamma c) in an IL-2R-reconstituted Ba/F3-derived cell line did not affect any biological responses including cell survival, disproving potential roles of the cleaved cytoplasmic domain of gamma c as a signal transducer. Collectively, these results indicated that potential nuclear function of cleaved type I cytokine receptor subunits is not plausible.