Yoshifumi Adachi

Reactive Oxygen Generated by NADPH Oxidase 1 (Nox1) Contributes to Cell Invasion by Regulating Matrix Metalloprotease-9 Production and Cell Migration

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKα kinase and the degradation of IκBα, suppressing the NFκB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity.

Parietal cell autoantigens involved in neonatal thymectomy-induced murine autoimmune gastritis. Studies using monoclonal autoantibodies

Autoimmune gastritis accompanied by autoantibodies to parietal cells was induced in BALB/c nu/+ mice by neonatal thymectomy 2-4 days after birth. Three monoclonal autoantibodies, designated as 2B6 (IgG1), 2G10 (IgG2b), and 1H9 (IgG1), were obtained from one of these mice. All three reacted specifically with parietal cells, 2G10 recognizing species-specific antigenic determinants and 2B6 and 1H9 recognizing interspecies-specific antigenic determinants. All three recognized antigens on the membrane of intracellular secretory canaliculi and the cytoplasmic tubulovesicular system of parietal cells. At least two different molecular groups were recognized by these antibodies; 2B6 recognizing a 65,000-79,000-mol wt group and 1H9 recognizing a 92,000-120,000-mol wt group. Sera of most mice with autoimmune gastritis reacted with either or both groups. Both groups were consistently coprecipitated by any of the three antibodies when solubilized in NP-40. Sera, from patients with pernicious anemia, containing anti-parietal cell antibodies could also precipitate these two groups of antigens. Competition assay and physicochemical studies showed that the epitopes recognized by the three monoclonal antibodies are different.

Efficient large-scale purification of non-histone chromosomal proteins HMG1 and HMG2 by using Polybuffer-exchanger PBE94.

A method for the efficient and practical large-scale purification of high-mobility group (HMG) non-histone chromosomal proteins, HMG1 and HMG2, from porcine thymus applying Polybuffer-exchanger PBE94 gel as anion-exchanger has been developed. This method affords higher resolution, purity and yield, than the conventional procedure of CM-Sephadex C-25 ion-exchange column chromatography. Furthermore, use of Polybuffer-exchanger PBE94 column chromatography led to direct preparation of HMG1 and HMG2 from loosely bound non-histone chromosomal protein fraction of chromatin without prefractional precipitation with trichloroacetic acid or prior extraction with perchloric acid. Thus, the application of PBE94 gel as an anion-exchanger to the subfractionation of other kinds of homologous protein is possible.

Cloning and characterization of LUN, a novel ring finger protein that is highly expressed in lung and specifically binds to a palindromic sequence.

We isolated cDNAs encoding a novel RING finger protein (LUN), the mRNAs of which were expressed at high levels in the lung. In situ hybridization revealed that LUN mRNAs were expressed in the alveolar epithelium of the lung. TheLUN gene locus was assigned to chromosome 9p21, which contains candidate tumor suppressor genes associated with loss of heterozygosity in more than 86% of small cell lung cancers. We clarified that LUN is localized to the nucleus and reveals Zn2+-dependent DNA binding activity. The region from amino acids 51 to 374 of LUN is responsible for DNA binding. Furthermore, we identified a novel palindromic binding consensus (5′-TCCCAGCACTTTGGGA-3′) for the LUN binding. Interestingly, this LUN binding palindromic sequence is found in the upstream transcriptional regulatory region of the E-cadherin gene and two intervening regions of the talin gene. Our results suggested that LUN might be an important trans-acting transcriptional regulator for lung cancer-associated genes including E-cadherin and talin genes.

A possible biochemical link between NADPH oxidase (Nox) 1 redox-signalling and ERp72.

Emerging evidence indicates that Nox (NADPH oxidase) 1-generated ROS (reactive oxygen species) play critical regulatory roles in various cellular processes, yet little is known of direct targets for the oxidase. In the present study we show that one of the proteins selectively oxidized in response to Nox1-generated ROS was ERp72 (endoplasmic reticulum protein 72 kDa) with TRX (thioredoxin) homology domains. Oxidation of ERp72 by Nox1 resulted in an inhibition of its reductase activity. EGF treatment of cells stimulated the Nox1 activity and the activated Nox1 subsequently mediated EGF-induced suppression of the ERp72 reductase activity. Co-immunoprecipitation, GST (glutathione transferase) pulldown assays and mutational analysis, indicated that Nox1 associates with ERp72, which involves its N-terminus encompassing a Ca(2+)-binding site and the first TRX-like motif. Furthermore, confocal microscopy showed co-localization between Nox1 and ERp72 at the plasma membrane. These results suggest that Nox1 functionally associates with ERp72, regulating redox-sensitive signalling pathways in a cellular context.

Protein kinase inhibitor H-7 blocks accumulation of unspliced mRNA of human T-cell leukemia virus type I (HTLV-I).

Rex, the post-transcriptional regulator of human T-cell leukemia virus type I (HTLV-I), is known to induce accumulation of the unspliced viral gag-pol mRNA. Rex is a phosphoprotein found in the cell nucleolus, whose function may be regulated by its localization and phosphorylation. We have examined the role of phosphorylation on Rex function by using a protein kinase inhibitor, H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine]. Treatment of an HTLV-I infected human T-cell line with H-7 blocked specifically accumulation of the unspliced gag-pol mRNA, resulting in the decreased Gag protein synthesis that corresponds with the decreased in vivo phosphorylation of Rex. In contrast, other viral and cellular products have not been influenced by the level of H-7 used. Therefore, the phosphorylation of Rex is required for the viral RNA partition of HTLV-I.

Ca2+-dependent cysteine proteinase, calpains I and II are not phosphorylated in vivo

To clarify phosphorylation of calpains I and II in vivo, we purified both calpains concurrently from the [32P] metabolic-labeled human chronic myelogenous leukemia cell line K-562. By Ultragel AcA34 column chromatography, enzymatic activity of calpain I was separated from [32P] radioactivity. Whereas calpain II activity was closely associated with [32P] radioactivity on Ultragel AcA34 and Blue Sepharose CL-6B column chromatographies. By the above purification procedures, calpain I was purified 1300-fold from the crude extract and calpain II was 920-fold from the original sample, respectively. Autoradiographies of purified calpains I and II from [32P] labeled K-562 cells revealed that both calpains were not specifically phosphorylated in vivo. The autophosphorylation in vitro on calpains and modulation of their proteolytic activities reported recently thus may not occur within cells.

The Calpain-Calpastatin System in Hematopoietic Cells

Calpain I requires low Ca2+ for activation and calpain II requires high Ca2+. It was generally accepted that erythrocytes contain calpain I and calpastatin, but no calpain II. We have recently found, however, that nucleated chicken erythrocytes contain both calpains I and II in addition to calpastatin. The finding is significant in rectifying the previous view that the chicken has only one molecular species of calpain, whereas mammals have two. Another erroneous view which prevailed previously was that polymorphonuclear (PMN) cells contain only one calpain species. We could also recently demonstrate that pig PMN cells do contain both calpains I and II. The cloning of cDNAs for calpastatin enabled us to utilize them as the probes in studying the expression of calpastatin in various hematopoietic cell-line cells. We found that several T cells infected with human retrovirus HTLV-I markedly increased the production of calpastatin, which could be measured both by calpain-inhibition assay and by Western blot analysis, but the level of mRNA for calpastatin did not significantly change when compared with noninfected T cells. The increase in calpastatin protein always parallels with the expression of interleukin 2 receptor protein by the HTLV-I-infected T cells, although the biological implication of such phenomena is almost entirely unknown yet.

Binding of cellular protein(s) to U3 region of human T-cell leukemia virus type-I long terminal repeat

The U3 region within the long terminal repeat (LTR) of human T-cell leukemia virus type-I (HTLV-I) contains elements responsible for transcriptionaltrans-activation. DNA-binding activities of nuclear proteins for each region of LTR were demonstrated using the gel retardation assay. The existence of cellular protein(s) specifically binding to the U3 region was demonstrated. Furthermore, the formation of the U3-protein(s) complex increased remarkably in HTLV-I-infected T-cells.

Expression of post-transcriptional regulatory gene of HTLV-I,rex, inEscherichia coli

Human T-cell leukemia virus type-I (HTLV-I) has a post-transcriptional regulatory gene termedrex. We have designed therex gene to express inE. coli. Synthesis ofrex protein, p27rex, was examined by immunoblot analysis using anti-p27rex antibody. No difference in electrophoretic mobility in NaDadSO4-PAGE was observed between p27rex expressed inE. coli and in an HTLV-I-infected cell line, MT-2. Slower migration of p27rex, corresponding to a 27-kD protein, in NaDodSO4-PAGE when compared with the calculated molecular weight from the amino acid sequence (Mr=20,367) is suggested to be caused not by posttranslational modification, but by the intrinsic nature of the protein, which is rich in proline and arginine.