Enhanced expression of Harvey ras induced by serum deprivation in cultured astrocytes.

Trophic deprivation contributes to astrocyte damage that oc- curs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to no- vel cytoprotective strategies. Cultured mouse astrocytes re- sponded to trophic deprivation with a large and transient increase in the expression of p21ras, 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 p21ras levels was largely attenuated by the reducing agent, N-ace- tylcysteine, 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 lev- els was sensitive to the protein synthesis inhibitor, cyclohex- The mechanisms underlying astrocyte damage are largely unexplored, although this process is relevant to the patho- physiology of acute and chronic neurodegenerative disorders (Bridges et al. 1992; Le Prince et al. 1995; Bruijn et al. 1997; Rafałowska and Podlecka 1998; Lee et al. 1999; Mouser et al. 2006). Unraveling these mechanisms might offer new targets for therapeutic intervention. Activation of P2Y1 receptors, for example, has been shown to protect astrocytes against oxidative damage (Shinozaki et al. 2005). We focused on the small monomeric G protein, p21ras, which is a key regulator of the signaling cascade triggered by oxidative stress (Cuda et al. 2002) and is also a target for reactive oxygen species (ROS) and cellular redox stress (Cheng and Meinkoth 2001). Ras-expressing cells produce imide, 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 protea- some 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 cul- tured 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 in- creased expression in Ha-Ras, which is limited by the con- comitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.