No abstract

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two‐hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity‐binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 ± 11 nM and 125 ± 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type‐1 repeat (TSP‐1) domain of CTGF binds to the exon 7‐coded region of VEGF165 and that the COOH‐terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full‐length CTGF and the deletion mutant possessing the TSP‐1 domain inhibited VEGF165‐induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein‐to‐protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

No abstract

Objective The goal of this study was to investigate the role of endogenous amyloid-β peptide (Aβ) in healthy brain. Methods Long-term potentiation (LTP), a type of synaptic plasticity that is thought to be associated with learning and memory, was examined through extracellular field recordings from the CA1 region of hippocampal slices, whereas behavioral techniques were used to assess contextual fear memory and reference memory. Amyloid precursor protein (APP) expression was reduced through small interfering RNA (siRNA) technique. Results We found that both antirodent Aβ antibody and siRNA against murine APP reduced LTP as well as contextual fear memory and reference memory. These effects were rescued by the addition of human Aβ42, suggesting that endogenously produced Aβ is needed for normal LTP and memory. Furthermore, the effect of endogenous Aβ on plasticity and memory was likely due to regulation of transmitter release, activation of α7-containing nicotinic acetylcholine receptors, and Aβ42 production. Interpretation Endogenous Aβ42 is a critical player in synaptic plasticity and memory within the normal central nervous system. This needs to be taken into consideration when designing therapies aiming at reducing Aβ levels to treat Alzheimer disease.

ADAMTS4 (aggrecanase-1) is considered to play a key role in the degradation of aggrecan in arthritides. The inhibitory activity of tissue inhibitors of metalloproteinases (TIMPs) to ADAMTS4 was examined in an assay using aggrecan substrate. Among the four TIMPs, TIMP-3 inhibited the activity most efficiently with an IC 50 value of 7.9 nM, which was at least 44-fold lower than that of TIMP-1 (350 nM) and TIMP-2 (420 nM) and s 250-fold less than that of TIMP-4 (2 W WM for 35% inhibition). These results suggest that TIMP-3 is a potent inhibitor against the aggrecanase activity of ADAMTS4 in vivo. ß

ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ⌬Sp (⌬Arg 693 -Lys 837 , lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC 50 ‫؍‬ 110 nM), whereas no inhibition was observed with ⌬Sp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC 50 ‫؍‬ 170 nM). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family are composed of at least 19 molecules (1) and are involved in various biological and biochemical events such as fertilization, proteoglycan degradation, processing of fibrillar collagens, and intravascular coagulation (2-7). Among them, ADAMTS1, ADAMTS4 (also referred to as aggrecanase-1), ADAMTS5 (aggrecanase-2), and ADAMTS9 cleave specific Glu-X bonds (where X is most often Ala or Gly) of the core protein of aggrecan, a major proteoglycan in articular cartilage (3,6,7). Previous studies suggested that the major aggrecan fragments found in vitro in response to cytokine-stimulated cartilage degradation and in vivo in arthritic joint fluids are generated through cleavage at Glu-X bonds by the glutamyl endopeptidase activity of these AD-AMTS members (8,9). Recent studies further demonstrated that ADAMTS4 is induced by stimulation of chondrocytes and synovial cells with interleukin-1, tumor necrosis factor-␣, or transforming growth factor-␤, although ADAMTS5 is constitutively expressed (6, 10). In addition, ADAMTS4 is overexpressed by synovial cells and chondrocytes in osteoarthritis and rheumatoid arthritis (10). Thus, ADAMTS4 is considered to play an important role in the aggrecan degradation of articular cartilage in osteoarthritis and rheumatoid arthritis. The aggrecanase...

We isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus (ϳ40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (ϳ19 kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, fibroblasts. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.

Matrix metalloproteinase-7 (MMP-7) (also known as matrilysin-1) is secreted as a proenzyme (proMMP-7) and plays a key role in the degradation of various extracellular matrix (ECM) and non-ECM molecules after activation. To identify the binding proteins related to proMMP-7 activation, a human lung cDNA library was screened by yeast two-hybrid system using proMMP-7 as bait. We identified a candidate molecule CD151, which is a member of the transmembrane 4 superfamily. Complex formation of proMMP-7 with CD151 was demonstrated by immunoprecipitation of the molecules from CaR-1 cells, a human rectal carcinoma cell line, expressing both proMMP-7 and CD151, and CD151 stable transfectants incubated with proMMP-7. Yeast twohybrid assays using deletion mutants of proMMP-7 and CD151 suggested an interaction between the propeptide of proMMP-7 and the COOH-terminal extracellular loop of CD151. The binding activity of 125 I-labeled proMMP-7 to CD151 on the cell membranes was shown with CD151 stable transfectants. Laser-scanning confocal microscopy demonstrated that proMMP-7 colocalizes with CD151 on the cell membranes of CD151 stable transfectants and CaR-1 cells. In situ zymography using crosslinked carboxymethylated transferrin, a substrate of MMP-7, demonstrated proteinase activity on and around CD151 stable transfectants and CaR-1 cells, while the activity was abolished by their treatment with MMP inhibitors, anti-MMP-7 antibody or anti-CD151 antibody. In human lung adenocarcinoma tissues, colocalization of MMP-7 and CD151 was demonstrated on the carcinoma cells. Metalloproteinase activity was present in these tissues and could be inhibited by antibodies to MMP-7 or CD151. These data demonstrate for the first time that proMMP-7 is captured and activated on the cell membranes through interaction with CD151, and suggest the possibility that similar to the MT1-MMP/MMP-2 system, MMP-7 is involved in the pericellular activation mechanism mediated by CD151, a crucial step in proteolysis on the cell membranes under various pathophysiological conditions including cancer invasion and metastasis.