Kazushige Maki

Role of cytosolic phospholipase A2 in allergic response and parturition

Phospholipase A2 (PLA2) comprises a superfamily of enzymes that hydrolyse the ester bond of phospholipids at the sn-2 position. Among the members of this superfamily, cytosolic PLA2 has attracted attention because it preferentially hydrolyses arachidonoyl phospholipids and is activated by submicromolar concentrations of Ca2+ ions and by phosphorylation by mitogen-activated protein kinases (MAP kinases). Here we investigate the function of cytosolic PLA2 in vivo by using homologous recombination to generate mice deficient in this enzyme. These mice showed a marked decrease in their production of eicosanoids and platelet-activating factor in peritoneal macrophages. Their ovalbumin-induced anaphylactic responses were significantly reduced, as was their bronchial reactivity to methacholine. Female mutant mice failed to deliver offspring, but these could be rescued by administration of a progesterone-receptor antagonist to the mother at term. Considered together with previous findings, our results indicate that cytosolic PLA2 plays a non-redundant role in allergic responses and reproductive physiology.

Defective B-1 Cell Development and Impaired Immunity against Angiostrongylus cantonensis in IL-5Rα-Deficient Mice

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G−/− homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF‐hC3G. Expression of Cre abolished the expression of C3G in MEF‐hC3G and inhibited cell adhesion‐induced activation of Rap1. The Cre‐expressing MEF‐hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R‐Ras. Expression of Epac and CalDAG‐GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G‐deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G‐dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

Transcriptional Regulation of the Mouse IL-7 Receptor α Promoter by Glucocorticoid Receptor

Expression of the IL-7R α-chain (IL-7Rα) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7Rα gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7Rα mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7Rα promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of ∼270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7Rα promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7Rα promoter. Thus, this study identifies the IL-7Rα CNS-1 region as a GC-responsive element.

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.

The bacteriophage P1 Cre/loxP site‐specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP‐flanked DNA sequences in a gene‐targeted locus in fertilized eggs. We replaced a part of the T‐cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP‐flanked neogene in mouse embryonic stem (ES) cells by gene‐targeting technique. The resulting ES cell clones containing the mutant allele (VγLNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild‐type female mice. One hundred and seventy‐six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS‐Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre‐mediated recombination, resulting in a deletion of the loxP‐flanked sequences (VγΔ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the VγΔ locus to their offspring. The other 8 pups carried only wild‐type alleles. There were no pups carrying the unrecombined VγLNL locus. Thus, the frequency of Cre‐mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS‐Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the VγLNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997.

Differential Roles of Cytokine Receptors in the Development of Epidermal γδ T Cells

IL-7 and IL-15 play important roles in γδ T cell development. These receptors transmit proliferation and/or survival signals in γδ T cells. In addition, the IL-7R promotes recombination and transcription in the TCR γ locus. To clarify the role of the cytokine receptors in the development of epidermal γδ T cells, we introduced a Vγ3/Vδ1 TCR transgene, derived from Thy-1+ dendritic epidermal T cells (DETC), into IL-7Rα-deficient mice, and we found that they partly rescued γδ T cells in the adult thymus but not in the spleen. Introduction of an additional Bcl-2 transgene had a minimal effect on γδ T cells in the adult thymus of these mice. In contrast to the adult thymus, the introduction of the Vγ3/Vδ1 TCR transgene into IL-7Rα−/− mice completely restored Vγ3+ T cells in the fetal thymus and DETC in the adult skin. On the contrary, the same Vγ3/Vδ1 TCR transgene failed to rescue DETC in the skin of IL-2Rβ-deficient mice, even with the additional Bcl-2 transgene. These results suggest that the IL-2/IL-15R, rather than the IL-7R, plays an essential role in proliferation and survival of DETC in the fetal thymus and the skin. In contrast, the IL-7R is probably essential in the induction of V-J recombination of TCRγ genes. Thus, this study proves that IL-7R and IL-2/IL-15R serve differential functions in epidermal γδ T cell development.

Role of Hepatocyte-Derived IL-7 in Maintenance of Intrahepatic NKT Cells and T Cells and Development of B Cells in Fetal Liver

The liver contains a variety of resident immune cells, such as NK cells, NKT cells, T cells, macrophages, and dendritic cells. However, little is known about how IL-7, which is produced by hepatocytes, functions locally in development and maintenance of liver immune cells. To address this question, we established IL-7–floxed mice and crossed them with albumin promoter-driven Cre (Alb-Cre) transgenic mice to establish conditional knockout of IL-7 in hepatocytes. The levels of IL-7 transcripts were reduced 10-fold in hepatocyte fraction. We found that the absolute numbers of NKT and T cells were significantly decreased in adult liver of IL-7f/f Alb-Cre mice compared with IL-7f/f control mice. In contrast, NK cells, dendritic cells, and B cells were unchanged in the IL-7f/f Alb-Cre liver. The number of Vα14+ invariant NKT cells was significantly reduced in liver, but not in thymus and spleen, of IL-7f/f Alb-Cre mice. Furthermore, B cell development was impaired in perinatal liver of IL-7f/f Alb-Cre mice. This study demonstrates that hepatocyte-derived IL-7 plays an indispensable role in maintenance of NKT and T cells in adult liver and development of B cells in fetal liver, and suggests that hepatocytes provide a unique IL-7 niche for intrahepatic lymphocytes.

MEK1/2 Induces STAT5-Mediated Germline Transcription of the TCRγ Locus in Response to IL-7R Signaling

The IL-7R plays an essential role in γδ T cell development by inducing V-J recombination of the TCRγ locus through STAT5. Although tyrosine residues in the intracellular domain of the mouse IL-7R α-chain (IL-7Rα) have been implicated in STAT5 activation, it is still unknown whether they are essential for γδ T cell development. In this study, we showed that those IL-7Rα tyrosine residues are not essential for γδ T cell development, because phenylalanine replacement of four intracellular tyrosine residues (IL-7R-FFFF) partially rescued γδ T cell development of IL-7Rα−/− progenitors. To examine signaling pathways activated by IL-7R-FFFF, we introduced a chimeric receptor consisting of the human IL-4R α-chain and mouse IL-7R-FFFF (4R/7R-FFFF) into an IL-7-dependent pre-B cell line and found that 4R/7R-FFFF induced TCRγ germline transcription and STAT5 activation. Treatment of cells with MEK1/2 inhibitors significantly decreased levels of TCRγ germline transcription and STAT5 tyrosine phosphorylation mediated by 4R/7R-FFFF, suggesting that MEK1/2 plays an alternative role in STAT5 activation by IL-7R. MEK1/2 associated with STAT5 and induced STAT5 tyrosine phosphorylation and DNA binding activity. Furthermore, MEK1 directly phosphorylated a STAT5 tyrosine residue in vitro. Finally, active MEK1 partially rescued TCRγ germline transcription by IL-7R in a pre-T cell line. These results demonstrate that MEK1/2 induces TCRγ germline transcription by phosphorylating STAT5 through IL-7R-FFFF and suggest a potential role for MAPK in IL-7R tyrosine-independent activation of STAT5.

Myeloid differentiation is impaired in transgenic mice with targeted expression of a dominant negative form of retinoid X receptor β

To investigate the in vivo function of retinoid X receptor (RXR) on myelopoiesis, we generated transgenic (Tg) mice with targeted expression of a dominant negative form of RXR β in myeloid cells. In these Tg mice the transgene is expected to suppress the function of heterodimeric receptors composed of RXR and its counterparts, such as retinoic acid receptor. Out of 12 mice analysed, one Tg mouse exhibited a severe maturation arrest at the promyelocytic stage. Three other Tg mice showed a mild inhibition of myeloid differentiation, which was further augmented when mice were treated with 5‐fluorouracil (5‐FU). Furthermore, four Tg mice showed impaired myeloid differentiation in response to the treatment by 5‐FU or granulocyte‐colony stimulating factor (G‐CSF), although they exhibited apparently normal myelopoiesis in the untreated state. The phenotype of Tg mice observed after G‐CSF treatment correlated with the expression level of the transgene, although the correlation was not found in untreated mice. These results indicated that myeloid differentiation is perturbed in the Tg mice by the dominant negative effect of the transgenic RXR, indicating that RXR plays a role in myelopoiesis.

Molecular cloning and characterization of TPP36 and its isoform TPP32, novel substrates of Abl tyrosine kinase

We have molecularly cloned TPP36, a novel 36 kDa protein with 281 amino acids that was identified as a protein phosphorylated in B progenitor cells following stimulation with pervanadate/H2O2. Analysis with anti‐TPP36 antiserum revealed that TPP36 was expressed ubiquitously and had an isoform with 236 amino acids, designated TPP32. TPP36/32 were localized mainly in cytoplasm despite the presence of a typical nuclear localization signal sequence. These proteins were phosphorylated preferentially by Abl among a panel of tyrosine kinases examined. Phosphorylation of tyrosine 120 in TPP36/32 led to an apparent mobility shift in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting conformational change in the phosphorylated protein. Thus, TPP36/32 appear to be novel substrates of Abl tyrosine kinase.