Abstract
Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Among mammalian tissues, the highest levels of p21Ras protein are detected in the brain. Here, we investigated the expression of KRAS and HRAS proto-oncogenes in primary astrocytes following acute oxidative stimulation. Reactive oxygen species (ROS) changed the expression of proto-oncogenes at both transcriptional and translational levels. De novo protein synthesis analysis measured approximate values of proteins half-life, ranging from 1–4 h, of the different H- and K- isoforms by western blot analysis. Quantitative gene expression analysis of KRAS and HRAS revealed an unexpected short-term induction of KRAS mRNA in primary astrocytes in response to acute stimulation. Indeed, cultured astrocytes responded to proteasomal inhibition by preventing the reduction of c-K-Ras. A fraction of K-Ras protein accumulated in the presence of ROS and cycloheximide, while a substantial proportion was continuously synthesized. These data indicate that ROS regulate in a complementary fashion p21Ras isoforms in primary astrocytes: K-Ras is rapidly and transiently induced by post-translational and post-transcriptional mechanisms, while H-Ras is stably induced by mRNA accumulation. We suggest that K-Ras and H-Ras are ROS sensors that adapt cells to metabolic needs and oxidative stress.
Highlights
The small guanine nucleotide binding proteins of the p21Ras family, encompassing in mammals the highly homologous H-Ras, N-Ras, and K-Ras isoforms, are actively expressed in mouse astrocytes [1,2,3,4]
Astrocytes—We studied the direct effects of hydrogen peroxide addition in regulating the protein levels of K-Ras and H-Ras in cultured astrocytes
Our experiments show that mild oxidative insults, as subtoxic H2 O2, strongly activate astrocytic Ras pathways by acute stimulation
Summary
The small guanine nucleotide binding proteins of the p21Ras family, encompassing in mammals the highly homologous H-Ras, N-Ras, and K-Ras isoforms, are actively expressed in mouse astrocytes [1,2,3,4]. Most of the studies on p21Ras signaling are based on enforced expression of p21Ras mutant protein [8,9,10,11,12], which, because of its mechanism of action cannot discriminate between contributions from different isoforms or from other members of the p21Ras superfamily. The monomeric GTP-binding protein, p21Ras , has an established role in activating these pathways and has been implicated in the cellular response to oxidative stress [24,25] and is a common signaling target of reactive free radicals and cellular redox stress [7]. How individual p21Ras proto-oncogenes KRAS and HRAS react to oxidative stimuli is still poorly understood This manuscript describes the expression of wild-type K-Ras and H-Ras in primary astrocytes following acute oxidative stimulation, with the reactive oxygen species playing a key role. ROS appears to affect the expression of isoforms (a) in discrete ways and (b) at both the transcriptional and translation levels
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