CD44 signaling through focal adhesion kinase and its anti-apoptotic effect

Adhesion molecules can initiate intracellular signaling. Engagement of CD44 either by its natural ligand hyaluronan or a specific antibody on a cell line induced tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which then associated with phosphatidylinositol 3-kinase (PI3K) and activated mitogen-activated protein kinase at its downstream. However, the introduction of dominant negative Rho into the cells inhibited the CD44-stimulated FAK phosphorylation. Cells expressing CD44 were significantly resistant to etoposide-induced apoptosis. This anti-apoptotic effect was cancelled by the inhibition of either Rho, FAK or PI3K. These results may indicate a signaling pathway from CD44 to mediate the resistance against drug-induced apoptosis in cancer cells.     

Cloning and biochemical characterization of astacin-like squid metalloprotease

We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and ALSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time.     

Hsp105alpha enhances stress-induced apoptosis but not necrosis in mouse embryonal f9 cells

Hsp105alpha, which belongs to the HSP105/110 family, is expressed at especially high levels in the brain in mammals and has been shown to prevent stress-induced apoptosis in neuronal cells. This protein is also expressed transiently at high levels during mouse embryogenesis, and is characteristically found in apoptotic cells and bodies in embryos. In the present study, to elucidate a role of Hsp105alpha in embryonal cells, we established Hsp105alpha-overexpressing F9 cells, and examined the effect of Hsp105alpha on cell death induced by etoposide, heat shock or cycloheximide. Apoptotic cell death was induced in cells treated with etoposide or heat shock, and necrotic cell death was induced in cells by cycloheximide. The apoptosis was enhanced by overexpression of HSP105alpha, whereas the necrosis was not affected by overexpression of HSP105alpha. Furthermore, Hsp105alpha seemed to modulate the stress-induced apoptosis at different steps of the apoptotic process depending on the stress stimuli. The present findings together with the previous observation on neuronal cells suggested that Hsp105 has opposite effects on stress-induced apoptosis depending on the cell type; a pro-apoptotic effect in embryonal cells and an anti-apoptotic effect in neuronal cells. The apoptosis-enhancing activity of Hsp105alpha may play an important role during embryogenesis.     

Nerve growth factor enhances neurotransmitter release from PC12 cells by increasing Ca(2+)-responsible secretory vesicles through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase

Neurotrophins play important roles in the differentiation and survival of neurons during development, and in the regulation of synaptic transmission in adult brain. Brief treatment with nerve growth factor (NGF) enhances depolarization and ionomycin-induced dopamine and acetylcholine release from PC12 cells. The enhancing effect appears very quickly and reaches a plateau 10-15 min after application. NGF also enhances hypertonic solution-induced dopamine release, and increases the amount of dopamine released from membrane-permeabilized PC12 cells in the absence of MgATP, suggesting that NGF enhances neurotransmitter release by increasing the number of Ca(2+)-responsive secretory vesicles. The activation of Trk receptors is essential for NGF action, since K252a abolishes the NGF-induced potentiation of dopamine release and brain-derived neurotrophic factor enhanced ionomycin-induced release only in TrkB-expressing cells. NGF-mediated potentiation of dopamine release is completely abolished by wortmannin, a PI 3-kinase inhibitor, and by U0126 and PD98059, MAP kinase kinase inhibitors, indicating that the activation of PI 3-kinase and MAP kinase pathways is essential for NGF action. These findings suggest that NGF regulates neurotransmitter release through the activation of TrkA receptors, possibly by increasing the number of secretory vesicles in a readily releasable pool.     

Specific molecular interactions of oversulfated chondroitin sulfate E with various heparin-binding growth factors. Implications as a physiological binding partner in the brain and other tissues

We previously observed that the cortical neuronal cell adhesion mediated by midkine (MK), a heparin (Hep)-binding growth factor, is specifically inhibited by oversulfated chondroitin sulfate-E (CS-E) (Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413) and that CS-E exhibits neurite outgrowth promoting activities toward embryonic rat hippocampal neurons. We have also shown oversulfated CS chains in embryonic chick and rat brains and demonstrated that the CS disaccharide composition changes during brain development. In view of these findings, here we tested the possibility of CS-E interacting with Hep-binding growth factors during development, using squid cartilage CS-E. The binding ability of Hep-binding growth factors (MK, pleiotrophin (PTN), fibroblast growth factor-1 (FGF-1), FGF-2, Hep-binding epidermal growth factor-like growth factor (HB-EGF), FGF-10, FGF-16, and FGF-18) toward [(3)H]CS-E was first tested by a filter binding assay, which demonstrated direct binding of all growth factors, except FGF-1, to CS-E. The bindings were characterized further in an Interaction Analysis system, where all of the growth factors, except FGF-1, gave concentration-dependent and specific bindings. The kinetic constants k(a), k(d), and K(d) suggested that MK, PTN, FGF-16, FGF-18, and HB-EGF bound strongly to CS-E, in comparable degrees to the binding to Hep, whereas the intensity of binding of FGF-2 and FGF-10 toward CS-E was lower than that for Hep. These findings suggest the possibility of CS-E being a binding partner, a coreceptor, or a genuine receptor for various Hep-binding growth factors in the brain and possibly also in other tissues.     

Fibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway

The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparin-like molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23. The present findings have revealed a novel MAPK-dependent mechanism of the regulation of phosphate uptake by FGF signaling.     

Hypermethylation and histone deacetylation lead to silencing of the maspin gene in human breast cancer

Serum transferrins are monomeric glycoproteins with a molecular mass of around 80 kDa, that transport iron to cells via receptor-mediated endocytosis. Although both serum transferrins (STfs) and ovotransferrins (OTfs) are derived from the same gene in aves, the ovotransferrins do not transport iron in vivo. Crystal structures of OTf have been solved, in contrast no three-dimensional structure of avian STf have been determined as yet. Here we report the purification, crystallization, and preliminary crystallographic studies of the hen STf both in apo- (iron free) and holo- (iron loaded) forms. The hen STf has been purified to homogeneity by hydrophobic interaction chromatography. Both the apo- and holo-forms were crystallized by hanging drop vapor diffusion method at 277 K. The apo-crystals diffract to a resolution of 3.0 A and belong to the space group P4(3)2(1)2 with unit cell parameters a=b=90.5 and c=177.9 A. The holo-crystals diffract to a resolution of 2.8 A and belong to space group P2(1) with a=72.8, b=59.6, c=88.2 A, and beta=95.7 degrees.     

Subgenomic replicon derived from a cell line infected with the hepatitis C virus

Recently, cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. To see whether a HCV sequence derived from HCV-infected cultured cell sequence can be used for the construction of a functional replicon, a HCV subgenomic RNA carrying a neomycin-resistant gene was constructed using the HCV genome RNA obtained from cultured cells infected with HCV. After transfection, G418-resistant Huh-7 cells were selected and subcloned. Finally, the production of HCV proteins and de novo synthesis of subgenomic RNA were confirmed in the selected cell clone, indicating that this subgenomic RNA replicated in cultured cells and functioned as a replicon. These results suggest that the HCV genome obtained from an in vitro HCV infection system with cultured cells can be used to develop a subgenomic replicon system with diverse HCV sequences.     

Atrophic change of rat salivary gland during adenovirus-induced hyperleptinemia

Sustained hyperleptinemia in normal rats induced by infusing a recombinant adenovirus containing the rat leptin cDNA (AdCMV-leptin) exhibited a remarkable reduction in food intake (AdCMV-leptin, 9.3 +/- 2.6 vs untreated, 20.6 +/- 1.0 g/day) and ablated body fat without any significant changes in wet weight of liver and left ventricle. In those hyperleptinemic rats, we found a 52% reduction in wet weight of salivary gland compared with that in the pair-fed AdCMV-beta-gal-treated rats, which received a recombinant virus containing the beta-galactosidase gene (AdCMV-beta-gal) and were fed on the same amount of food as had been consumed by the AdCMV-leptin-treated group on the previous day. Microscopic examination with hematoxylin-eosin staining revealed that atrophic change was induced in both serous and mucous gland only in the AdCMV-leptin-treated group, but not in the pair-fed controls. Thus, the atrophic changes in hyperleptinemic rats were due to neither a decrease of food intake nor disuse of the salivary gland related with anorexia. Our data suggested that size of the salivary gland was controlled, at lease in part, by "non-anorexic" effect of leptin.