title: Enhanced expression of Harvey ras induced by serum deprivation in cultured astrocytes.
abstract: Trophic deprivation contributes to astrocyte damage that occurs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to novel cytoprotective strategies. Cultured mouse astrocytes responded to trophic deprivation with a large and transient increase in the expression of p21(ras), which was secondary to an enhanced formation of reactive oxygen species (ROS) detected by cytofluorimetric analysis after preloading with 2',7'-dichlorofluorescein diacetate. The increase in p21(ras) levels was largely attenuated by the reducing agent, N-acetylcysteine, which was proven to reduce ROS formation in astrocytes subjected to serum deprivation. We extended the analysis to the Ha-Ras isoform, which has been implicated in mechanisms of cytotoxicity. We found that serum deprivation enhanced the expression and activity of Ha-Ras without changing Ha-Ras mRNA levels. The increase in Ha-Ras levels was sensitive to the protein synthesis inhibitor, cycloheximide, suggesting that serum deprivation increases translation of preformed Ha-Ras mRNA. The late decline in Ha-Ras levels observed after 60 min was prevented by the proteasome inhibitor, MG132, as well as by the selective mitogen-activated protein kinase (MAPK) inhibitor, PD98059. Serum deprivation led to the activation of the MAPK pathway in cultured astrocytes, as shown by an increase in phosphorylated extracellular signal-regulated kinase 1/2 levels after 5 and 30 min. Finally, using the siRNA technology, we found that an acute knock-down of Ha-Ras was protective against astrocyte damage induced by serum deprivation. We conclude that cultured astrocytes respond to trophic deprivation with an increased expression in Ha-Ras, which is limited by the concomitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.
authors.affiliation: Department of Experimental Medicine and Pathology, University of Rome 'La Sapienza', Rome, Italy. samantha.messina@uniroma1.it
authors.forename: Samantha
authors.initials: S
authors.lastname: Messina
authors.forename: Gemma
authors.initials: G
authors.lastname: Molinaro
authors.forename: Valeria
authors.initials: V
authors.lastname: Bruno
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Battaglia
authors.forename: Paola
authors.initials: P
authors.lastname: Spinsanti
authors.forename: Alba
authors.initials: A
authors.lastname: Di Pardo
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Nicoletti
authors.forename: Luigi
authors.initials: L
authors.lastname: Frati
authors.forename: Antonio
authors.initials: A
authors.lastname: Porcellini
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors
chemical_list: C110400:2',7'-dichlorodihydrofluorescein diacetate; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D003513:Cycloheximide; D015689:Oncogene Protein p21(ras); C022616:thiazolyl blue