Toshio Heike

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRβ) and gp130. We reported that the intracellular domain of gp130 plays an important role in self‐renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self‐renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self‐renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4‐hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.

Tool-use learning induces BDNF expression in a selective portion of monkey anterior parietal cortex

Learning but not execution of tool-use induced expression of brain-derived neurotrophic factor (BDNF). The expression was highest in the anterior bank of the intraparietal sulcus, especially in the region posteriorly adjacent to the somatosensory shoulder and forearm region in area 3b, suggesting that BDNF plays a role in altering the body image of the hand to include the repeatedly used tool as its extension.

Tool-use learning selectively induces expression of brain-derived neurotrophic factor, its receptor trkB, and neurotrophin 3 in the intraparietal multisensorycortex of monkeys.

When humans repeatedly use a tool, our body image alters until the tool finally becomes a part or an extension of the body. This alteration of body image perhaps results from re-integration of somatosensory and visual signals. We trained Japanese monkeys to use a rake-shaped tool to retrieve a distant food pellet, then used a novel tissue-sampling method to suction brain tissue from the anterior bank of their intraparietal sulcus, where somatosensory and visual signals converge. Examination of the messenger RNA expression levels of neurotrophins and their receptors using real-time quantitative polymerase chain reaction revealed learning-selective induction in the expression of brain-derived neurotrophic factor, its receptor trkB, and NT-3 during, but not after, the learning. These results suggest that these factors are involved in the reorganization of the somatosensory and visual signals in the anterior bank of the intraparietal sulcus when monkeys are learning the use of the tool.

Bovine papilloma virus encoded E2 protein activates lymphokine genes through DNA elements, distinct from the consensus motif, in the long control region of its own genome.

Activation of T cells by antigen, lectin or a combination of phorbol ester (PMA) and calcium ionophore (A23187) leads to the induction of a set of lymphokine genes. Transfection of a human T cell leukemia cell line, Jurkat, or an African green monkey kidney cell line, CV1, with a cDNA encoding E2 protein, a trans‐activator of bovine papilloma virus type 1, results in activation of interleukin 2 (IL‐2), interleukin 3 (IL‐3) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) genes in a transient transfection assay. 5′ deletion and mutation analyses showed that the sequence between positions −60 and a TATA‐like sequence is required for basic promotor function and that the sequence between positions −95 and −73 containing conserved lymphokine element 2 (CLE2) and a GC box (CLE2/GC box) mediates the positive response to E2 protein. The latter has been previously shown to respond to PMA/A23187 stimulation or to p40tax, a trans‐activator encoded by human T cell leukemia virus type 1 (HTLV‐I). The sequence located between −108 and −99 (CLE1) is inhibitory to E2 protein or PMA/A23187 stimulation. The combination of E2 protein and PMA/A23187 appears to eliminate an inhibitory effect of the upstream region. However, E2 protein, like p40tax, mediates a positive response through CLE1 alone linked to the basic promoter sequence. The level of activation of the long control region (LCR) by E2 protein is unaffected by the number of CLE2/GC box sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of lymphokine genes in T cells: Role of cis-acting DNA elements that respond to T cell activation signals

Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Th1 and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines. The production of the granulocyte-macrophage colony stimulating factor (GM-CSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions -95 to +27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein(s) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLEO defines a binding site for protein(s) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-1, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation.

Hypomethylation of the proximal and intronic regulatory regions of the IFN-γ gene is not essential for its transcription by naive CD4+ T cells cultured with IL-4

Recently, long-term preculture with IL-4 or IL-7 has been reported to induce IFN-gamma-producing ability in naive CD4+ T cells without stimulation via TCR. The mechanism of IFN-gamma-transcription in naive CD4+ T cells precultured with IL-4 was analyzed and compared with that in typical Th1 cells by focusing on the TATA proximal and first intronic regulatory regions of the IFN-gamma gene. Both regulatory regions in these IL-4-primed naive CD4+ T cells, which produce a large amount of IFN-gamma upon stimulation with PMA and ionomycin, were completely methylated in contrast to the same hypomethylated regions in Th1 cells. DNase I hypersensitive site analysis suggested that both regulatory regions in IL-4-primed naive CD4+ T cells were not active for IFN-gamma-expression. Moreover, we demonstrated that the composition of transcriptional factors that can bind to the proximal regulatory region is different between IL-4-primed naive CD4+ T cells and Th1 cells. These results indicated that the transcriptional machinery involved in the expression of the IFN-gamma gene by CD4+ T cells varied depending on their modes of differentiation in both the responsive regulatory regions and the specific nuclear factors.

X-linked severe combined immunodeficiency with γδT cells

A patient with X‐linked severe combined immunodeficiency (X‐SCID) was found to have a deletion mutation of a four base pair in the transmembrane domain of the IL‐2 receptor γ chain gene, a subunit shared by the receptors for IL‐4, IL‐7, IL‐9, and IL‐15 (common γ chain; γc). He had very few αβT cells but had a considerable number of γδT cells in his peripheral blood. Fluorescence in situ hybridization (FISH) analysis showed that the γδT cells in his peripheral blood were not of maternal origin. He had received a Bacillus Calmette‐Guerin (BCG) vaccination before recognition of the disease, and the BCG infection remained quiescent with no reaction for 19 months. After successful bone marrow transplantation, the site of the BCG vaccination showed a reaction, and live BCG were detected. It is useful to consider the relationship between the existence of γδT cells and BCG in this case, and it is suggested that γδT cells may be, in a given situation, less dependent on the γc chain than are αβT cells.

The activation of gp130/LIFR stimulates the proliferation of neural stem cell

The self-rcncwal mechanism of mammmalian brain neural stem cell has well been studied until recently, and one of them is Ras-MAPK signal pathway activated by EGF and bFGF. But littlc is known about the role of JAK-Tyk family signal oathwav in neural stem cell. In this studv the influence of LIFR/eo130 activation on neural stem cell oroliferation was Examined. From the transgcnic mouse embryo expressing huma;;‘GM-CSFR extracellular domain aid LIFR/gpl30 intracellular domain. striatal neural stem cell was prepared. After that human GM-CSF was added and the effect on neural stem cell was ana,lyzcd. It was shown that human GM-CSF promoted the proliferation of neural stem cell with doscdcpcndcncy, but its mitogenic activity was wcakcr than that of mouse EGF. And neural stem cell differentiated into neuron, astrocytc and oligodcndrocytc without human GM-CSF. In this study, the conclusion was that the JAK-Tyk family signal pathway via LIFRigpl30 was involved in brain neural stem cell self-renewal mechanism. The existcncc of another signal pathway involved in neural stem ccl1 proliferation implies its different role in nerve injury or regeneration.

T Cell Activation Signals and Regulation of Lymphokine Gene by Viral and Cellular Transactivators

T cells activated by antigen stimulation produce a set of lymphokines. By employing protein kinase C (PKC) which is active without stimulation or viral transactivator HTLV-I p40tax or BPV E2 protein, we characterized the T cell antigen receptor signal transduction pathway downstream of PKC. Consistent with the earlier observations that activation of PKC and Ca2+ influx are necessary for T cell activation, the IL-2 promoter is activated by actions of constitutively active PKC and Ca2+ ionophore in the human T cell leukemia line Jurkat. We found that p40tax or E2 protein activate transfected GM-CSF gene as well as SV40 and HIV promoters without external stimuli. The sequence of GM-CSF promoter required for stimulation by PMA/A23187 is localized between positions -95 and -73 (CLE2). The same region responds to p40tax or E2 protein. Another sequence, located between -113 and -96 (CLE1), mediates inducible response to p40tax but not to E2 protein or PMA/A23187 stimulation. Activation of the SV40 promoter by p40tax or E2 protein is dependent on SV40 enhancer sequences. Only one copy of the segment carrying the NF-κ B binding site is sufficient to mediate the induction by E2 protein, p40tax or PMA/A23187 stimulation. HIV LTR promoter also responds to E2 protein or p40tax through the same DNA element. These results indicate that p40tax or E2 protein activate GM-CSF and viral promoters by interacting with cellular component(s) in the T cell activation signal transduction pathway.