Kouji Kuno

ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function.

A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.

ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan

A disintegrin‐like and metalloproteinase with thrombospondin type I motifs‐1 (ADAMTS‐1) is an extracellular matrix‐anchored metalloproteinase. In this study we have demonstrated that ADAMTS‐1 is able to cleave a major cartilage proteoglycan, aggrecan. N‐terminal sequencing analysis of the cleavage product revealed that ADAMTS‐1 cleaves the Glu1871–Leu1872 bond within the chondroitin sulfate attachment domain of aggrecan. In addition, deletional analysis demonstrated that the C‐terminal spacer region of ADAMTS‐1 is necessary to degrade aggrecan. These results suggest that ADAMTS‐1 may be involved in the turnover of aggrecan in vivo.

The IL‐1 receptor signaling pathway

Interleukin‐1 (IL‐1) exerts pleiotropic effects on a variety of tissues through binding to its receptor. Two distinct types of receptors for IL‐1 have been characterized in mouse and human. Most of the IL‐1 signal has been shown to be transmitted through type I IL‐1R (80 kDa) in T lymphocytes as well as B lymphocytes and monocytes. Type II receptor may act as a suppressor of IL‐1 biological activities by competing in binding with type I receptors on the cell surface. Functional studies of the type I IL‐1R demonstrated that the cytoplasmic segments, possessing a sequence similarity with the Drosophila Toll gene product or IL‐6R β chain, gpl30, are important for transmitting activity that induces cytokine genes. In the past three years, several groups reported that IL‐1 and tumor necrosis factor (TNF) rapidly induce sphingomyelin turnover in various types of cells, producing ceramide, which may act as a second messenger molecule in an intracellular signaling cascade. Activation of both acid and neutral sphingomyelinases (SMases) has been suggested, and Schutz et al. proposed that the phosphatidylcholine–phospholipase C/acid SMase pathway is involved in TNF‐induced NF‐xB activation. However, our recent study showed that the NF‐xB activation is induced by IL‐1/TNF in fibroblasts from patients with type A Niemann‐Pick disease, with acid SMase deficiency. This finding implies that acid SMase activity is not essential for the activation of NF‐xB by IL‐1/TNF at least in fibroblasts. Other signaling pathways including neutral SMase and unidentified protein kinases may be important for NF‐xB–mediated cytokine gene activation. J. Leukoc. Biol. 56: 542–547; 1994.

Expression of both types of human interleukin-8 receptors on mature neutrophils, monocytes, and natural killer cells.

cDNA cloning revealed the presence of two related but distinct types of human interleukin‐8 (IL‐8) receptors, type I (type A) and type II (type B). By immunizing rabbits with glutathione‐S‐transferase fused with the NH2‐terminal domain of each type of IL‐8 receptor, we prepared polyclonal antibodies that specifically recognized the NH2‐terminal domain of each type of IL‐8 receptor. Immunofluorescence analysis of human peripheral blood leukocytes demonstrated that mature granulocytes except eosinophils express both types of IL‐8 receptors. A majority of monocytes and CD16+ natural killer (NK) cells in peripheral blood were stained with both antibodies, whereas CD3+ T or CD20+ B lymphocytes in peripheral blood or CD34+ cells in cord blood were not stained with either antibody. These results suggest that both types of human IL‐8 receptors were coordinately and selectively expressed in mature granulocytes, monocytes, and CD16+ NK cells. J. Leukoc. Biol. 57: 180–187; 1995.

Stimulation of Interleukin-8 Production by Okadaic Acid and Vanadate in a Human Promyelocyte Cell Line, an HL-60 Subline POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE ON THE OKADAIC ACID-INDUCED NF-κB ACTIVATION

Most types of cells can produce interleukin (IL)-8 in response to various inflammatory stimuli. To study the role of protein phosphatases in the signal transduction leading to IL-8 production, a subline of HL-60 (C-15) was treated with okadaic acid (OA) and sodium orthovanadate (VA), inhibitors of phosphoserine/phosphothreonine phosphatase and phosphotyrosine phosphatase, respectively. Both OA and VA dramatically increased IL-8 secretion up to 200-fold in the HL-60 cells. OA and VA stimulation was accompanied by a marked increase in IL-8 mRNA expression and also by activation of a transcription factor, NF-κB. In addition, an essential role of the NF-κB site in the IL-8 gene activation was confirmed by the chloramphenicol acetyltransferase assay. IL-8 production by OA or VA was inhibited by protein kinase inhibitors, including staurosporine, H-7, K252a, herbimycin A, and genistein. Both OA and VA induced significant tyrosine phosphorylation of p44, which was presumed to be Erk1, a member of the mitogen-activated protein kinase family, with concomitant activation of the mitogen-activated protein kinase activity. In parallel, rapid degradation of IκB-α, an inhibitory component of NF-κB, was observed. Since OA-activated Erk1 phosphorylated recombinant IκB-αin vitro, we assumed that Erk1 is involved in the phosphorylation and subsequent degradation of IκB-α, thus leading to the activation of IL-8 gene transcription.

Novel insight into molecular mechanism of endotoxin shock: biochemical analysis of LPS receptor signaling in a cell-free system targeting NF-kappaB and regulation of cytokine production/action through beta2 integrin in vivo.

Lipopolysaccharide (LPS), a constituent of gram‐negative bacteria cell wall, plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as cytokines, nitrous oxide, superoxide anions, and lipid mediators. In vitro, LPS induces the transcription of a set of genes involved in inflammatory reactions by activating several types of transcription factors, particularly nuclear factor‐κB (NF‐κB). An analysis of NF‐κB activation using a cell‐free system demonstrated that two pathways converge to activate NF‐κB; one is staurosporine‐sensitive, the other is staurosporine‐insensitive and tyrosine kinase‐dependent. Furthermore, the latter pathway culminates in IκBaL phosphorylation at serine/threonine residues in its carboxyl‐terminal acidic region with dissociation of IκBα from NF‐κB, thereby activating NF‐κB. The requirement for the phosphorylation at this site was confirmed by the specific inhibition of NF‐κB activation in a cell‐free system by the synthetic peptide corresponding to this site. The in vivo administration of an anti‐CD18 antibody prevented elevation of plasma tumor necrosis factor (TNF) levels and acute lethality induced by injection of a low dose of LPS into Propionibacterium acnes‐primed rabbits or by the administration of a single high dose of LPS into animals. Anti‐CD18 also prevented acute lethality induced by one of the main mediators of endotoxin shock, TNF‐α. Furthermore, an antibody to a ligand for CD18, intercellular adhesion molecule‐1, also prevented TNF‐induced shock as well as endotoxin shock in rabbits. These observations suggest that the interaction between leukoytes and endothelium through β2. integrin adhesion molecules may be of primary importance in mediating LPS signals in vivo.

Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice.

Blocking monoclonal antibodies (mAbs) specific to mouse interleukin‐1 receptor antagonist (IL‐1ra) were prepared by immunizing Armenian hamsters with recombinant mouse IL‐1ra. A sensitive and specific ELISA against mouse IL‐1ra was also established. In Propionibacterium αcnes‐induced liver injury, P. acnes induced transient increase of serum tumor necrosis factor‐α levels but not those of IL‐1ra, IL‐1, and IL‐6. However, subsequent lipopolysaccharide (LPS) challenge induced the increase of serum levels of all these cytokines and the peak serum IL‐1ra level was more than 20 times as high as serum IL‐1 levels. Immunohistochemical analysis demonstrated that IL‐1ra was predominantly produced by hepatocytes during the course of the priming phase by P. acnes and eliciting phase by LPS challenge. Furthermore, the administration of a mAb to mouse IL‐1ra exacerbates the liver injury induced by P. acnes and sublethal dose of LPS, suggesting a protective role of endogenous IL‐1ra in this liver injury model. J. Leukoc. Biol. 58: 90–98; 1995.

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

AbstractPurpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein lα (hu-MIP-lα), murine-macrophage inflammatory protein lα (mu-MIP-lα), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-lα, mu-MIP-lα, or hu-IL-8 expression vector. The production of hu-MIP-1α reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 105 cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-lα, mu-MIP-lα, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-lα and mu-MIP-lα. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-lα showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-lα gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-lα gene might be useful as an effective therapy for the treatment of certain tumors.

Human MCAF Gene Transfer Enhances the Metastatic Capacity of a Mouse Cachectic Adenocarcinoma Cell Line in Vivo

AbstractPurpose. To evaluate the effect of monocyte chemotactic and activating factor (MCAF/MCP-1/JE) on tumor progression and metastasis. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCAF expression vector. Spontaneous lung metastases were determined in mouse. Results. The production of MCAF reached 0.4 ng/ml in vitro when transfectant cells were cultured at a cell density of 5 × 104 cells/ml for 3 days. Transfection of MCAF expression vector did not affect the growth rate in vitro. Also, after MCAF-transfection, the size of tumors after intra-footpad inoculation was similar to that of the parental cells. When the primary tumors were resected on the 10th day after inoculation, the incidence of spontaneous lung metastasis was less than 20% in both cells. The number of endothelial cells in the primary tumor rapidly increased from the 10th to the 14th day after inoculation, as revealed by immunohistochemical staining. In accordance with enhanced angiogenesis, the incidence rates of spontaneous metastasis increased when the primary tumors were resected on the 14th day after inoculation. Moreover, the spontaneous lung metastases were augmented in the animals injected with MCAF-transfectants compared to those injected with parental cells with a concomitant increase of angiogenesis. Conclusions. These results suggest that MCAF may augment the metastatic potential by modulating tumor associated angiogenesis.

Cloning of a cDNA encoding a mouse homolog of the interleukin-8 receptor

A mouse cDNA library was screened using a DNA fragment generated by polymerase chain reaction (PCR) with oligodeoxyribonucleotide primers which were derived from the conserved sequences in cDNAs encoding the human and rabbit interleukin-8 receptors (hIL-8R and rIL-8R). A novel cDNA was obtained encoding 359 amino acids (aa) with seven putative transmembrane portions similar to hIL-8R and rIL-8R. Its aa sequence shows 64 and 69% homology to those of type-1 and type-2 hIL-8R, respectively. COS-7 cells transfected with the isolated cDNA in a mammalian expression vector bind IL-8, but do not bind a related protein, monocyte chemotactic and activating factor, suggesting that the isolated cDNA encodes the mouse homolog of IL-8R. Northern blot analysis showed that mRNA of this clone was highly expressed in mouse peritoneal neutrophils, and the single band was observed in Southern blotting analysis on mouse genomic DNA digested with HindIII or KpnI, suggesting that this is a single-copy gene.