Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs.
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We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs. (+info)
Structure and promoter activity of the human glia maturation factor-gamma gene: a TATA-less, GC-rich and bidirectional promoter.
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Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity. (+info)
Glia maturation factor produced by thymic epithelial cells plays a role in T cell differentiation in the thymic microenvironment.
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In order to determine molecules on thymic epithelial cells which play an important role in the process of T cell differentiation, mAb recognizing thymic epithelial cells were made using stromal cells of the embryonic thymus at 15-gestation date. Among many mAb, a specific one was selected in terms of its inhibition of T cell development in an in vitro culture system of the embryonic thymus. cDNA of the protein recognized by one of the mAb was obtained by a panning method. Sequence analysis revealed that the protein was identical to glia maturation factor (GMF)-beta. Northern blot analysis confirmed the expression of GMF-beta mRNA in the thymus and brain. Furthermore, immunoblotting analysis identified the production of GMF-beta protein in the thymus, the brain and a thymic epithelial cell line. GMF-beta protein prepared by a glutathione-S-transferase gene fusion system greatly influenced T cell development in the in vitro culture system of the embryonic thymus in favor of a significant increase of CD4(+) T cells with expression of TCRbeta. These data taken together suggest that GMF-beta protein is produced by thymic epithelial cells and plays a role in T cell development in favor of CD4(+) T cells. (+info)
Induction of glia maturation factor-beta in proximal tubular cells leads to vulnerability to oxidative injury through the p38 pathway and changes in antioxidant enzyme activities.
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Proteinuria is an independent risk factor for progression of renal diseases. Glia maturation factor-beta (GMF-beta), a 17-kDa brain-specific protein originally purified as a neurotrophic factor from brain, was induced in renal proximal tubular (PT) cells by proteinuria. To examine the role of GMF-beta in PT cells, we constructed PT cell lines continuously expressing GMF-beta. The PT cells overexpressing GMF-beta acquired susceptibility to cell death upon stimulation with tumor necrosis factor-alpha and angiotensin II, both of which are reported to cause oxidative stress. GMF-beta overexpression also promoted oxidative insults by H2O2, leading to the reorganization of F-actin as well as apoptosis in non-brain cells (not only PT cells, but also NIH 3T3 cells). The measurement of intracellular reactive oxygen species in the GMF-beta-overexpressing cells showed a sustained increase in H2O2 in response to tumor necrosis factor-alpha, angiotensin II, and H2O2 stimuli. The sustained increase in H2O2 was caused by an increase in the activity of the H2O2-producing enzyme copper/zinc-superoxide dismutase, a decrease in the activities of the H2O2-reducing enzymes catalase and glutathione peroxidase, and a depletion of the content of the cellular glutathione peroxidase substrate GSH. The p38 pathway was significantly involved in the sustained oxidative stress to the cells. Taken together, the alteration of the antioxidant enzyme activities, in particular the peroxide-scavenging deficit, underlies the susceptibility to cell death in GMF-beta-overexpressing cells. In conclusion, we suggest that the proteinuria induction of GMF-beta in renal PT cells may play a critical role in the progression of renal diseases by enhancing oxidative injuries. (+info)
Mitogen-expanded Schwann cells retain the capacity to myelinate regenerating axons after transplantation into rat sciatic nerve.
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We have developed a method for genetically modifying Schwann cells (SCs) in vitro and then assessed whether these SCs could interact normally with axons in vivo. Rat SCs were transduced in vitro with the lacZ gene by using a retroviral vector and then expanded with the SC mitogens forskolin and glial growth factor. These mitogen-expanded SCs had an abnormal phenotype as compared to both SCs in vivo and primary SCs in vitro, yet when they were introduced into a regenerating rat sciatic nerve, they formed morphologically normal myelin sheaths around the axons. These results demonstrate that SCs can be genetically altered, their numbers expanded in culture, and yet respond appropriately to axonal signals in the peripheral nervous system. This approach offers a plausible way to manipulate genes involved in axon-SC interactions, including genes that may be defective in some inherited peripheral neuropathies. (+info)
Sensitive immunoassays for human and rat GMFB and GMFG, tissue distribution and age-related changes.
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We developed sensitive and specific two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) using specific antibodies raised in rabbits. These assay systems enabled us to identify GMFB and GMFG (GMFs) in both human and rat samples and they were used to investigate the tissue distribution and serum concentrations of human and rat GMFs. In the case of rat, relatively high levels of GMFB were found in the central nervous system, except for the spinal cord, and in thymus and colon. Higher levels of GMFG were found in the thymus, spleen and colon. The distribution of GMFs in human was similar to that in rat. In the rat, the maximum serum concentration of GMFG was at 4 weeks of age. The decrease in its level was rapid for the first 30 days of life in both sexes. On the other hand, the concentration of GMFB in serum did not change significantly with age. Similarly, in human, the concentration of GMFG in serum was highest in the 21-30-year-old group and began to decrease rapidly in the 30-year-old group. In contrast, the concentration of GMFB did not change significantly during this period. No significant sex differences in the serum levels of GMFs were observed in human and rat. The present EIA systems are sufficiently sensitive for studying GMFs in human and rat organs. (+info)
Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.
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Actin cytoskeleton reorganization is a fundamental process for actin-based cellular functions such as cytokinesis, phagocytosis, and chemotaxis. Regulating actin cytoskeleton reorganization is therefore an attractive approach to control endothelial and inflammatory cells function and to treat cardiovascular diseases. Here, we identified glia maturation factor-gamma (GMFG) as a novel factor in actin cytoskeleton reorganization and is expressed preferentially in microvascular endothelial and inflammatory cells. During mouse embryogenesis, GMFG was expressed predominantly in blood islands of the yolk sac, where endothelial and hematopoietic cells develop simultaneously. In endothelial cells, GMFG was colocalized with F-actin in membrane ruffles and was associated with F-actin assessed by actin co-sedimentation assay. Interestingly, GMFG was phosphorylated at N-terminal serine, and its phosphorylation was enhanced by coexpression of dominant active Rac1 and Cdc42. Furthermore, a pseudophosphorylated form of GMFG (GMFG-S2E) demonstrated higher association with F-actin. Stable expression of GMFG-S2E remarkably enhanced stimulus-responsive lamellipodia and subsequent membrane ruffle formation in HeLa cells presumably through its interaction with Arp2/3 complex. Expression of GMFG enhanced actin-based cellular functions such as migration and tube-formation in endothelial cells. Moreover, we found that GMFG expression was significantly increased in a cardiac ischemia/reperfusion model where inflammation and angiogenesis take place actively. Taken together, our findings define a novel pathway in the regulation of actin-based cellular functions. Regulating GMFG function may provide a novel approach to modulate the pathophysiology of cardiovascular diseases. (+info)
A growth factor from neuronal cell lines stimulates myelin protein synthesis in mammalian brain.
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Oligodendroglia growth factor (OGF) is a 16-kDa soluble protein produced by neuronal cell lines. This factor, when incubated with brain glia in culture, selectively stimulates growth of oligodendroglia, the myelin-producing cells of the CNS. OGF infused into the cerebral cortex of the adult rat accelerates the production of myelin proteins as shown by increased specific activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase), by stimulated synthesis of myelin basic protein, and by elevations in levels of myelin proteolipid protein RNA. The ability of OGF to induce myelin protein production in vivo suggests that neuron-secreted growth factors help to regulate myelin formation within the CNS. (+info)