Cloning of a Novel RNA Binding Polypeptide (RA301) Induced by Hypoxia/Reoxygenation

Noriyuki Matsuo,Satoshi Ogawa,Yuji Imai,Tsutomu Takagi,Masaya Tohyama,David Stern,Akio Wanaka

doi:10.1074/jbc.270.47.28216

Abstract

Astrocytes have a critical role in the neuronal response to ischemia, as their production of neurotrophic mediators can favorably impact on the extreme sensitivity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed two ribonucleoprotein domains and serine/arginine-rich domains, suggestive of their function as RNA splicing factor. Northern analysis displayed striking induction only in cultured astrocytes within 15 min of reoxygenation and reached a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrated expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/reoxygenated astrocytes. Induction of RA301 mRNA was mediated, in large part, by endogenously generated reactive oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase which blocks oxygen-free radical formation by astrocytes. Similarly, increased expression of RA301 in supporting a neurotrophic function of astrocytes was suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expression is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues.

Highlights

Tissue injury consequent on ischemia results from two distinct types of mechanisms: first, adaptive processes to meet the challenges of oxygen deprivation and reduction in blood flow are marshalled, and second, the response to restoration of oxygenated blood flow, a central feature of which is the generation of oxygen-free radicals by a range of cells, is triggered
One common denominator of cellular mechanisms activated by hypoxia alone or hypoxia followed by reoxygenation is redirection of cellular biosynthetic processes leading to synthesis of new proteins and, changes in the cellular phenotype
Viability of Astrocytes Exposed to Hypoxia/Reoxygenation— Oxygen tension in the medium fell to 8 torr within 3–5 h after cultures were transferred to the hypoxia chamber

Summary

Introduction

Tissue injury consequent on ischemia results from two distinct types of mechanisms: first, adaptive processes to meet the challenges of oxygen deprivation and reduction in blood flow are marshalled, and second, the response to restoration of oxygenated blood flow, a central feature of which is the generation of oxygen-free radicals by a range of cells, is triggered. Astrocytes, the most abundant cell type in the central nervous system, are relatively resistant to the environmental stress imposed by hypoxia/reoxygenation, maintaining their capacity to proliferate and to generate neurotrophic mediators [5, 6]. This contrasts with the relative inability of neurons to maintain viability in response to such environmental perturbations. The identification of a putative RNA binding protein, with potential properties of a splicing factor based on analysis of the deduced amino acid sequence and whose expression is induced by hypoxia/reoxygenation, is likely to provide new insights into the cellular biosynthetic response to ischemia

Methods

Results

Conclusion