Akihide Takeuchi

The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage‐ and tissue‐specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34−/− mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34−/− MEF, although following treatment with tunicamycin, the eIF2α phosphorylation level did not change in either GADD34+/+ or GADD34−/− cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild‐type mice. These expressions were strongly reduced in GADD34−/− MEF, which suggests that GADD34 up‐regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148+/− mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148+/− tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148+/− embryonic stem cells and in fetal germ cells from chimeric Zfp148+/− embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

Microglial NO induces delayed neuronal death following acute injury in the striatum

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5‐μL ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti‐iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA‐I‐B4, ED‐1 and OX‐42. Haematoxylin‐eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl‐transferase‐mediated dUTP‐biotin nick end‐labelling (TUNEL) assay on the first day after the injury. These TUNEL‐positive neurons gradually disappeared toward the third day, while microglial cells increased. L‐Ng‐nitro‐arginine methylester (L‐NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.

Cloning and Characterization of a Transcription Factor That Binds to the Proximal Promoters of the Two Mouse Type I Collagen Genes

We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between −180 and −136 in the pro-α2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the −180 to −152 segment of the mouse pro-α2(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-α1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BFCOL1 participates in the transcriptional control of the two type I collagen genes.

In vitro and in vivo expression of inducible nitric oxide synthase during experimental endotoxemia: Involvement of other cytokines

In this study, we investigated the expression of genes for inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF‐α), interleukin 1β (IL‐1β), interleukin 6 (IL‐6) of Kupffer cells in the presence of lipopolysaccharide (LPS), and the tissue expression of iNOS in a rat liver after LPS injection at various time intervals. The effects of L‐NG‐nitroarginine‐methyl‐esther HCI (L‐NAMF), a NO inhibitor, also were examined. The mRNA transcripts of TNF‐α IL‐1β and IL‐6 were rapidly detected no more than at 1 h after LPS stimulation, whereas the iNOS transcript was detectable from 3 h after LPS stimulation and maximally increased at 12 h. This fact suggested that these early induced cytokines were related to expression of iNOS. Using an anti‐iNOS antiserum raised against recombinant iNOS protein, immunohistochemical analysis was made to reveal kinetics of NO producing cells. The cells immunoreactive for iNOS appeared at 6 h post‐LPS injection in the sinusoids of the liver. By structural and immunohistochemical studies, almost all iNOS positive cells were identified as Kupffer cells and endothelial cells. The number of cells immunoreactive for iNOS increased until 12 h post‐LPS injection. At 24 h after LPS injection, iNOS positive cells were restricted to the foci of spotty necrosis. Hepatic injury measured by released enzymes was increased by pretreatment of L‐NAME before LPS injection. J. Cell. Biochem. 65:349–358.

Cooperative interaction of NF-κB and C/EBP binding sites is necessary for manganese superoxide dismutase gene transcription mediated by lipopolysaccharide and interferon-γ

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by pro‐inflammatory cytokines. We investigated the cis‐acting elements within a tumor necrosis factor‐responsive element (TNFRE) which was identified in the second intron of the murine Mn‐SOD gene. Site‐directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF‐κB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis‐acting elements may be required for induction of Mn‐SOD gene expression mediated by lipopolysaccharide and interferon‐γ.

Macrophage colony-stimulating factor is expressed in neuron and microglia after focal brain injury.

In a previous study, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed neuronal death owing to the cytotoxic effect of microglial nitric oxide (NO), and these neurons are finally eliminated by activated microglia. In this process, microglia are activated to release NO, increase in number, and accumulate toward the damaged area. In this study, we investigated the expression of macrophage colony‐stimulating factor (M‐CSF, also called colony stimulating factor‐1; CSF‐1) and other cytokines, which are reported to relate to activation, proliferation, or migration of microglia. The mRNA of M‐CSF arose biphasically from 30 min to 1 hr and from 6 to 72 hr after the injury, as demonstrated by semiquantitative RT‐PCR. However, another cytokine of granulocyte‐macrophage CSF (GM‐CSF) or interleukin‐3 (IL‐3), which causes proliferation of microglia in vitro, was not detected. From immunohistochemical studies, positive staining of M‐CSF was observed mainly in neuron‐specific enolase (NSE)‐positive cells from 1 to 12 hr after the injury, and after that M‐CSF became positive in Griffonia simplicifolia isolectin‐B4 (GSA‐I‐B4)‐positive cells from 24 to 72 hr in the boundary area around necrosis. These results suggest that neurons around the damaged area express M‐CSF in the early phase after injury, which may initially activate microglia, and these activated microglia also express M‐CSF later, causing further proliferation or migration of microglia themselves to eliminate damaged neurons or necrotic brain tissue. J. Neurosci. Res. 65:38–44, 2001.

Cu/Zn- and Mn-superoxide dismutases are specifically up-regulated in neurons after focal brain injury.

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury.

Differential display analysis of murine collagen‐induced arthritis: cloning of the cDNA‐encoding murine ATPase inhibitor

We used the differential display technique in order to detect a new gene involved in murine type II collagen‐induced arthritis (CIA). In this study, we have identified a novel gene, IF1, whose expression level is increased during the natural course of CIA. Northern blot analyses suggest that IF1 is involved in the natural course of CIA but is not involved as a trigger of CIA. IF1 is considered to be the murine ATPase inhibitor gene for several reasons. First, IF1 shows an extremely high homology to the rat ATPase inhibitor; the highly conserved region between rat and bovine amino acid residues 22–45, which is the minimum sequence showing ATPase inhibitory activities, is also highly conserved in IF1. Second, IF1 possesses a histidine‐rich region in the same area, which is thought to be important for regulation of mammalian inhibitors. Third, the tissue distribution of IF1 is very suggestive. The expression of IF1 was very strong in energetic organs such as the heart, brain and kidney, and the development of arthritis requires great amounts of ATP. As arthritis develops rapidly, the cellular ATP pool may be decreased. Before the ATP pool is exhausted, the ATPase inhibitor may serve as a brake for ATP hydrolysis. If the supply of free energy can be reduced, the inflammation of arthritis may in turn be restored. Our hypothesis is that the ATPase inhibitor is involved in regulating the inflammatory responses.

1216 Microglial NO induces delayed neuronal death following acute injury in the striatum

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5-microL ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti-iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA-I-B4, ED-1 and OX-42. Haematoxylin-eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay on the first day after the injury. These TUNEL-positive neurons gradually disappeared toward the third day, while microglial cells increased. L-Ng-nitro-arginine methylester (L-NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.