Shinzo Nishi

Vav1/2/3-null mice define an essential role for Vav family proteins in lymphocyte development and activation but a differential requirement in MAPK signaling in T and B cells.

The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late “transitional” stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)–induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho–guanine nucleotide exchange factors.

Differential Requirements for Vav Proteins in DAP10- and ITAM-mediated NK Cell Cytotoxicity

Natural killer (NK) cells express multiple activating receptors that initiate signaling cascades through DAP10- or immunoreceptor tyrosine-based activation motif–containing adapters, including DAP12 and FcRγ. Among downstream signaling mediators, the guanine nucleotide exchange factor Vav1 carries out a key role in activation. However, whether Vav1 regulates only some or all NK cell–activating pathways is matter of debate. It is also possible that two other Vav family molecules, Vav2 and Vav3, are involved in NK cell activation. Here, we examine the relative contribution of each of these exchange factors to NK cell–mediated cytotoxicity using mice lacking one, two, or all three Vav proteins. We found that Vav1 deficiency is sufficient to disrupt DAP10-mediated cytotoxicity, whereas lack of Vav2 and Vav3 profoundly impairs FcRγ- and DAP12-mediated cytotoxicity. Our results provide evidence that these three Vav proteins function specifically in distinct pathways that trigger NK cell cytotoxicity.

Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis.

The rat GST-P (placental glutathione S-transferase), a phase II detoxifying enzyme, is not expressed in normal liver cells, but is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells. Results of previous studies indicated that GST-P gene activation was mainly controlled by an enhancer element, GPE1 (GST-P enhancer 1), but the specific activation mechanism of the GST-P gene was not fully understood [Morimura, Suzuki, Hochi, Yuki, Nomura, Kitagawa, Nagatsu, Imagawa and Muramatsu (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, Imagawa, Hirabayashi, Yuki, Hisatake, Nomura, Kitagawa and Muramatsu (1995) Cancer Res. 55, 2651-2655]. In the present study, we investigate the transcription factor Nrf2/MafK heterodimer (where Nrf2 stands for NF-E2 p45-related factor 2) as an activator of the GST-P gene through the action of GPE1 during hepatocarcinogenesis. Electrophoretic mobility-shift assay and footprinting analysis with wild-type GPE1 and GPE1 point mutants showed that the Nrf2/MafK heterodimer specifically bound GPE1. Reporter transfection assays indicated that Nrf2 strongly stimulated GST-P gene expression in mouse F9 embryonal carcinoma cells and H4IIE rat hepatoma cells. Northern-blot analysis indicated that GST-P and Nrf2 mRNA increased in parallel with development of precancerous lesions and hepatocellular carcinoma. Keap1 (Kelch-like ECH-associated protein 1), an inhibitory factor of Nrf2, decreased the activation of GPE1 by Nrf2 and this suppression was restored after treatment with electrophilic compounds. GST-P mRNA expression in H4IIE cells was induced by electrophilic compounds, as was the expression of mRNAs of other phase II detoxifying enzymes. Chromatin immunoprecipitation analyses showed that antibodies both against Nrf2 and against MafK precipitated GPE1 from the chromatin of the pre-neoplastic hepatocytes and rat hepatoma cells (H4IIE and dRLh84), but not from normal hepatocytes. These results indicate that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells.

Purification of human, dog and rabbit α-fetoprotein by immunoadsorbents of sepharose coupled with anti-human α-fetoprotein

A procedure is described for purification of human α-fetoprotein by an immuno-adsorbent column of Sepharose coupled with the antibody to human α-fetoprotein by means of BrCN. The purified α-fetoprotein was crystallized in (NH4)2SO4 solution. Dog and rabbit α-fetoprotein, which are immunologically cross-reactive with the antibody to human α-fetoprotein, were also purified by the antibody column to human α-fetoprotein.

Rat maf related genes: specific expression in chondrocytes, lens and spinal cord

maf is a family of oncogenes originally identified from avian oncogenic retrovirus, AS42, encoding a nuclear bZip transcription factor. We have isolated two maf related cDNA clones, maf-1 and maf-2, from a rat liver cDNA library. Comparison of the sequence homologies of the proteins encoded by maf-1 and maf-2 with those of c-maf and chicken mafB indicated that maf-1 and maf-2 are the rat homologues of mafB and c-maf, respectively. Both genes are expressed at low levels in a wide variety of rat tissues, including spleen, kidney, muscle and liver. Immunohistochemical studies and in situ hybridization analyses show that maf-1 and maf-2 are strongly expressed in the late stages of chondrocyte development in the femur epiphysis and the rib and limb cartilage of 15 day old (E15) embryo in rat. Cartilage cells, induced by subcutaneous implantation of bone morphogenic protein, also expressed maf-1 and maf-2. In situ hybridization analyses of E15 embryos show that both genes are expressed in the eye lens and the spinal cord as well as the cartilage. However, the expression patterns of maf-1 and maf-2 in lens and spinal cord are different.

Activation of mouse Pi-class glutathione S-transferase gene by Nrf2 (NF-E2-related factor 2) and androgen

The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions -59, -915 and -937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500 bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.

The structure and physicochemical properties of rat liver macrophage migration inhibitory factor.

We expressed rat macrophage migration inhibitory factor (rMIF) in E. coli using the cDNA isolated from a rat liver cDNA library. rMIF specifically bound glutathione (dissociation constant = 500 microM). We purified rMIF homogeneously on SDS-PAGE by S-hexylglutathione Sepharose affinity column chromatography and Sephadex G-100 column chromatography. The amino-acid sequence of rMIF was highly homologous to that of human MIF from a T-cell line; only a single amino-acid residue was substituted if conservative amino-acid substitutions were involved. The molecular weight of rMIF was calculated to be 12.4 kDa and 23.6 kDa by SDS-PAGE and analytical ultracentrifugation, respectively. Thus, it was concluded that the native rMIF formed a homodimeric structure. Proton nuclear magnetic resonance (1H-NMR) study revealed that rMIF was less thermostable (the denaturing temperature was from 50-60 degrees C) than human MIF (the denaturing temperature is about 80 degrees C (Nishihira et al. (1993) Biochem. Mol. Biol. Int. 31, 841-850). The secondary structure of rMIF evaluated by 1H-NMR experiments revealed that the contents of alpha-helix, beta-strand, and coil were 13.8%, 55.6%, and 30.6%, respectively.

Characterization of cysteine residues of glutathione S-transferase P : Evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47

Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.

IMMUNOLOGICAL AND CHEMICAL CORRELATION BETWEEN α-FETOPROTEINS FROM HUMAN AND SEVERAL MAMMALIAN SPECIES

Alpha-Fetoproteins of several animals were purified and their molecular weights, amino acid compositions and peptide maps were compared, demonstrating the close similarities. These data indicated that the alpha-fetoproteins of mammalian species have closely related antigenical and chemical structures. Rabbits and horses were immunized with human alpha-fetoprotein, and it was observed that the animals produced antibodies reaction not only with human alpha-fetoprotein but with their homologous alpha-fetoproteins. The results were interpreted as the breakdown of the tolerance to their own alpha-fetoprotein.

Cloning of the cDNA encoding the mouse ATBF1 transcription factor

We have isolated a mouse ATBF1 cDNA which is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis showed that levels of ATBF1 transcripts were low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation.