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ABSTRACT: We previously reported that clusterin enhances astrocyte proliferation and extracellular signal-regulated kinase (ERK) activity. It, however, remains largely unknown how clusterin promotes cell growth. Here, we investigate the signaling pathway and related molecules underlying astrocyte proliferation by clusterin. Exogenous clusterin stimulates Ras-dependent Raf-1/mitogen-activated protein kinase kinase (MEK)/ERK activation. Clusterin-induced astrocyte proliferation and ERK1/2 phosphorylation were abrogated by either AG1478 (an inhibitor of epidermal growth factor receptor, EGFR) or EGFR small interfering RNA. Furthermore, clusterin treatment provoked tyrosine phosphorylation of EGFR (pY(1173)), which was also blocked by AG1478. These results suggest that clusterin requires EGFR activation to deliver its mitogenic signal through the Ras/Raf-1/MEK/ERK signaling cascade in astrocytes.
Neuroreport 03/2009; 20(4):435-9. · 1.66 Impact Factor
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ABSTRACT: Clusterin, a secretory glycoprotein, has been shown to be up-regulated in the reactive astrocytes in response to brain injury and neurodegenerative diseases, but its function has not been clearly elucidated. In this study, we investigate whether clusterin has growth-stimulatory activity in astrocytes. Suppression of clusterin with antisense oligonucleotide induced growth arrest, whereas transient overexpression of clusterin by cDNA transfection or exogenous treatment with purified clusterin promoted proliferation of the primary astrocytes in culture. This clusterin-stimulated proliferation was abrogated by PD98059, an inhibitor of mitogen-activated protein kinase kinase. These results suggest that clusterin might play an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway.
Neuroreport 01/2007; 17(18):1871-5. · 1.66 Impact Factor
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ABSTRACT: Clusterin has been known as a chaperone-like molecule capable of interacting with various proteins. In this study, we show that clusterin interacts with the microtubule-destabilizing stathmin family protein SCLIP by GST pull-down and co-immunoprecipitation assays. Interestingly, SCLIP interacts with 80 kDa mature form of clusterin in the cytosolic fraction of PC12 cells permeabilized by low concentration of a weak nonionic detergent digitonin, but not with intracellular variants of clusterin known as binding isoforms of Ku70 or TGF-beta receptors. Both clusterin and SCLIP are co-localized at the perinuclear region and growth cone of PC12 cells. In addition, we show that the minimal domains for the interaction are mapped to the C-terminal valine-rich region (367-447) of clusterin and the N-terminal palmitoylation and membrane attachment site (1-34) of SCLIP. Finally, we demonstrate that ectopic expression of clusterin in PC12 cells elongates neurite-formation triggered by NGF and induces spontaneous neurite outgrowth even in the absence of NGF. Taken together, these results suggest that the clusterin interacts with SCLIP and the interaction may act as an important modulator during neuronal differentiation.
Experimental Cell Research 11/2005; 309(2):305-15. · 3.58 Impact Factor
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ABSTRACT: Since arginine deiminase (ADI; EC 3.5.3.6) inhibits cell proliferation by arresting cells in the G1 phase, we tested its synergistic effect on cell death induced by dexamethasone (DEX), which also induces apoptosis by G1 cell cycle arrest. ADI inhibited cell proliferation and induced apoptosis in human leukemic CEM cells in a dose-dependent manner. Simultaneous treatment with ADI and DEX showed synergistic effects on DNA fragmentation and LDH release. In addition, ADI exerted its anti-proliferative activity against DEX-resistant CEM cells. ADI suppressed expression of c-myc, a potential key regulator of cell proliferation and apoptosis, and increased expression of p27Kip1 cyclin-dependent kinase inhibitor. These results suggest that ADI efficiently increases the anti-cancer effect of DEX on human leukemic CEM cells through G1 cell cycle arrest involving downregulation of c-myc and upregulation of p27Kip1.
International Journal of Cancer 12/2004; 112(3):502-8. · 5.44 Impact Factor
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ABSTRACT: We have previously reported upregulation of clusterin at the time of islet cell regeneration after beta-cell injury. This led us to speculate that clusterin might be involved in the neogenic regeneration of the pancreas. Clusterin expression was examined throughout the process of pancreatic neogenesis in pancreatectomized rats. For in vitro analysis, duct cells were isolated from the rat pancreas and clusterin cDNA was transfected for its overexpression. Clusterin and its mRNA increased significantly in the early phase of regeneration, particularly at 1-3 days after pancreatectomy. Clusterin was transiently expressed in the differentiating acinar cells but faded afterwards. Interestingly, these clusterin cells were negative for PCNA (proliferating cell nuclear antigen), whereas most epithelial cells in ductules in the regenerating tissue showed extensive proliferative activity. Clusterin expression was also detected in some endocrine cells of the regenerating tissue. Transfection of clusterin cDNA into primary cultured duct cells resulted in a 2.5-fold increase in cell proliferation and induced transformation of non-differentiated duct cells into differentiated cells displaying cytokeratin immunoreactivity. Taken together, these results suggest that clusterin may play essential roles in the neogenic regeneration of pancreatic tissue by stimulating proliferation and differentiation of duct cells.
Journal of Histochemistry and Cytochemistry 11/2003; 51(10):1355-65. · 2.72 Impact Factor
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ABSTRACT: We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma arginine deiminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370+/-50 microM. Its optimal temperature and pH were 41 degrees C and pH 6.4, respectively, and enzyme activity remained > or = 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the endotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.
Molecules and Cells 02/2002; 13(1):137-43. · 2.18 Impact Factor
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ABSTRACT: Primary mixed glial cell cultures treated with lipopolysaccharide (LPS; 1.0 μg/ml) showed biphasic increases of inducible nitric oxide synthase (iNOS) mRNA expression 6 h and 24–36 h after LPS treatment. Dibutyryl-guanosine 5′,3′-cyclic monophosphate (db-cGMP; 1.0 mM) enhanced the second phase of the LPS-induced iNOS expression 24 and 30 h after LPS stimulation. KT5823 (1.0 μM), a protein kinase G (PKG) inhibitor, inhibited the LPS-induced iNOS expressions at 24 and 30 h and their enhancements caused by db-cGMP. In astrocyte-enriched cultures with reduced microglial contamination, the LPS-induced iNOS expression was decreased, though slightly enhanced by db-cGMP. These results suggest that cGMP/PKG signaling may be involved in the second phase of the LPS-induced glial iNOS expression and its upregulation, which are apparent in the presence of microglial cells.
Neuroscience Letters.
