(Received for publication, July 6, 1995; and in revised form, September 11, 1995)
From the
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.
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. Characteristics of the cellular response to
oxygen deprivation include redirection of energy metabolism, increased
glucose utilization, and expression of oxygen-regulated proteins (1, 2, 3) . During reoxygenation, distinct
mechanisms are triggered, such as generation of oxygen-free radicals,
and expression of heat shock proteins (HSP) ()and
glucose-regulated proteins, such as GRP78(4) . 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, thus,
changes in the cellular phenotype.
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. As we previously found that inhibition of protein synthesis during early reoxygenation prevented effective astrocyte adaptation to hypoxia/reoxygenation, resulting in eventual cell death(7) , we hypothesized that early during reexposure of cells to ambient oxygen critical gene products were expressed.
In this study we report the cloning of a novel putative RNA binding protein (RA301) based on differential display, which is strongly induced in cultured astrocytes early during reoxygenation, as well as in ischemic rat brain. Furthermore, inhibition of RA301 expression with a specific antisense probe reduced about 3-fold of release of interleukin-6 (IL-6), a neuroprotective cytokine, by astrocytes subjected to hypoxia/reoxygenation. 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.
Figure 1: Predicted amino acid sequence of RA301 (A), homology to Drosophila splicing regulator Tra-2 (B), and comparison with other RNA splicing regulators (C). The amino acid sequence of RA301 (288 residues) was predicted from a cDNA sequence. Serine/arginine-rich domain and RNA recognition motif (RRM) are indicated by bold italic type and are underline, respectively. Consensus sequences of ribonucleoprotein (RNP) sites are indicated by bold letters (Panel A). In Panel B, the RA301 sequence (upper line) is compared with the Tra-2 sequence, and an identical amino acid residue is indicated by an asterisk. The structure of RA301 is also compared with that of other RNA splicing regulators (Tra-2, SC35, and SF2/ASF). The serine/arginine-rich domain, RNP consensus sequence, and glycine-containing domain are indicated by a box with slant lines, filled box, and box with cross lines, respectively (Panel C).
Figure 2:
Induction of RA301 message in cultured rat
astrocytes by hypoxia/reoxygenation (A, Northern blot analysis
in pooled RNAs; B, time course; and C, effect of
cycloheximide and DPI). In Panel A, RNA was extracted and
pooled from three different cultures. About 5 µg of RNA from either
normoxic (lane N), hypoxic (lane H), or reoxygenated (lane H/R) cultures were subjected to Northern blot
hybridization using the RA301 cDNA probe (upper panel) and
-actin probe (lower panel) radiolabeled with
[
P]dCTP. In Panel B, total RNA was
extracted from astrocytes at the end of hypoxia (0 min after
reoxygenation) and the indicated time points (0.5-12 h after
reoxygenation). Then, about 5 µg of total RNA were subjected to
Northern blot analysis as indicated above. In Panel C, either
cycloheximide (10 µg/ml, Cx) or DPI (50 µM)
was added to the culture 15 min before reoxygenation, and total RNA was
applied to Northern blot analysis. The same analysis using RNAs
obtained at the end of hypoxia (lane H/R, 0), 15 and
60 min after reoxygenation (H/R, 15 and H/R, 60) from the same batch of experiments is also
shown.
Addition of cycloheximide (10 µg/ml) to
hypoxic astrocytes simultaneously with their placement in normoxia did
not block induction of RA301 mRNA (Fig. 2C), whereas
this concentration of cycloheximide blocked incorporation of
[H]leucine into material precipitable in
trichloroacetic acid by >90%. In contrast, the neutrophil-type NADPH
oxidase inhibitor, DPI, when present at the start of reoxygenation,
completely blocked the appearance of RA301 mRNA (Fig. 2C). Under these conditions, DPI prevents
generation of oxygen-free radicals by reoxygenated astrocytes based on
chemiluminescence using lucigenin as the substrate and the cytochrome c reduction assay(6) . Since DPI can also inhibit the
nitric oxide pathway, experiments were performed in which L-arginine-free medium was employed, or the competitive
inhibitor of nitric oxide synthase L-NMMA was present.
However, neither of these agents had a similar effect to DPI on RA301
mRNA (data not shown). These data suggested that oxygen-free radicals
formed early during the period of reoxygenation were likely to trigger
increased expression of RA301 mRNA, rather than induction of a
polypeptide requiring de novo protein biosynthesis.
Induction of RA301 in astrocytes exposed to hypoxia followed by
reoxygenation was not part of a generalized event reflecting
up-regulation of multiple RNA binding factors and related proteins. By
Northern analysis, U1-70K, U2AF, and U2AF
did
not display increased expression at the mRNA level, compared with the
results previously obtained with RA301 (Fig. 3).
Figure 3:
Effect of hypoxia/reoxygenation on the
other mammalian RNA binding factors and related peptides. Astrocytes
(about 1 10
cells) were exposed to normoxia,
hypoxia (24 h), and hypoxia/reoxygenation (1 h followed by
reoxygenation). Total RNA extracted from each culture (lanes
N, H, and H/R, respectively) was then subjected
to Northern blot analysis using radiolabeled U1-70K (Panel A),
U2AF
(Panel B), and U2AF
(Panel
C) probes. In each autoradiogram,
-actin was employed as an
internal control (Panel D).
Figure 4:
Induction of RA301 antigen in cultured rat
astrocytes: time course (A) and effect of antisense
oligonucleotide on the hypoxia/reoxygenation-mediated induction of
RA301 (B) and HSP72 (C). Astrocytes plated on
7.5-cm dishes were exposed to either hypoxia (HYPO) alone or hypoxia/reoxygenation (REOXY). Cells
were then harvested at the indicated time point (0-24 h in the
hypoxia chamber and 2-24 h after reoxygenation) and subjected to
Western blotting using anti-RA301 antiserum (Panel A). In Panels B and C, either cycloheximide (10 µg/ml, CX), YS-60 antisense oligonucleotide (10 µM, ANTI), or sense oligonucleotide (10 µM, SENSE) was added to the culture 30 min before reoxygenation.
Cells were harvested 3 h after reoxygenation and subjected to Western
blotting using either anti-RA301 antiserum (Panel B) and
monoclonal antibody to HSP72 (Panel C). In Panel C, HYPO and REOX.3HR denote the samples obtained from
hypoxic (24 h) and reoxygenated (3 h) cultures, respectively. The
migration of standard proteins is indicated on the far left side of the gel: trypsin inhibitor (21.5 kDa), carbonic anhydrase (31
kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (97.4 kDa),
-galactosidase (116 kDa), and myosin (200
kDa).
Figure 5: Induction of RA301 message (Panels A, B, D, and E) and antigen (Panel C) in ischemic rat brain. Brain ischemia was introduced in rats by the unilateral occlusion of middle cerebral artery. 12 and 24 h after a 2-h ischemic insult, rats were sacrificed, and brain slices were subjected to in situ hybridization using the RA301 cRNA probe. Note the up-regulation of RA301 message in the ischemic hemisphere (A and B). Microautoradiogram of Panel B shows diffusely up-regulated message for RA301 (D). Panel E shows a higher magnification of Panel D (bracketed area). The most superficial layer (layer 1) of the cerebral cortex (indicated by arrows) which contains few neurons was heavily labeled, suggesting that astrocytes express up-regulated RA301 transcripts. In Panel C, brain tissue was cut out from either a ischemic lesion or a control nonischemic cortex 24 h after the ischemic event. Protein was then extracted from each sample and subjected to Western blotting using the anti-RA301 antiserum.
Figure 6:
Effect of RA301 antisense oligonucleotide
on hypoxia/reoxygenation-mediated induction of IL-6: time course (A) and effects of other oligonucleotide sequences (B). Astrocytes planted on 24-well plate (about 6
10
cells/well) were exposed to hypoxia for 32 h and
reoxygenated with or without the addition of antisense oligonucleotide
(YS-60; 5`-GCT GTC GCT CAT GAC TCG GG-3`). IL-6 activity in culture
supernatant was then assessed at the indicated time point by MH-60
proliferation assay (Panel A). Three other structures of
antisense phosphorothioate oligonucleotide corresponding to sense
structure to YS-60 (Sense), 5`-GCC GCT GTC GCT CAT GAC TC-3` (YS-59), and 5`-GTC GCT CAT GAC TCC GGG TT-3` (YS-61)
were added to the culture 30 min before reoxygenation, followed by the
IL-6 activity assay of the culture supernatant 8 h after reoxygenation (Panel B). In each panel mean ± S.D. are shown (**p < 0.05 by multiple comparison).
Exposure of cells to a period of oxygen deprivation followed
by reoxygenation imposes a major metabolic stress. The period of
hypoxia, in which there is a shift to anaerobic glycolysis, is
associated with events such as up-regulation of the
non-insulin-dependent glucose transporter(2) , activation of
NF-IL-6, and transcription of target genes, including IL-6 and tumor
necrosis factor-(22) , which we have hypothesized primes
the cells for the subsequent phase of reoxygenation. It is also likely
that other events, such as activation of AP-1, which is stimulated by
antioxidants, will occur during hypoxia, further modifying biosynthetic
mechanisms(23, 24) . In contrast, during
reoxygenation, generation of oxygen-free radicals occurs. This is
especially striking in vivo when leukocytes are attracted to
reperfused tissues and activated, and their formation of reactive
oxygen intermediates is induced. Previous studies have drawn attention
to a role for oxygen-free radicals in triggering production of
polypeptide mediators relevant to the biology of ischemia; reactive
oxygen intermediates appear to initiate expression of IL-1 and IL-8 in
mononuclear phagocytes (25, 26) and to induce
synthesis and elaboration of IL-6 in astrocytes(6) . One
mechanism through which such reactive oxygen species impact on the
biosynthetic apparatus is through activation of the transcription
factor NF-kB(27) , thereby accelerating the rate of
transcription of particular mRNAs. Another means for coordinately
regulating expression of protein products is through processing of
multiple RNA species.
Our findings demonstrate that a novel 36-kDa polypeptide, RA301, is induced by oxygen-free radicals produced endogenously by cultured astrocytes exposed to hypoxia followed by reoxygenation. Based on the deduced amino acid sequence, domains characteristic of RNA binding/splicing proteins are present, including two serine/arginine-rich motifs, an RNA recognition motif, and a glycine-rich motif. RA301 bears closest homology to Tra-2, an RNA splicing factor important in sex determination in Drosophila(28) . Thus, RNA splicing factors, such a Tra-2, can have a fundamental role in altering cellular phenotype by maturation of nascent RNAs. Further studies will be required to prove that RA301 also has properties of an RNA splicing factor.
The presence of RA301 in ischemic regions of rat brain further emphasizes its potential expression in loci where it is likely to impact on ultimate expression of proteins in cellular elements subject to ischemia. The importance of such de novo protein products in the cellular adaptation to hypoxia/reoxygenation is illustrated by the induction of cell death which invariably follows addition of cycloheximide to reoxygenated astrocytes following 8 h of reoxygenation (7) . These studies provide a first step in characterizing a gene product of potential importance in the cellular response to ischemia, RA301, and raise multiple questions concerning its potential impact on redirecting essential biosynthetic events in astrocytes, and, potentially, other cells exposed to hypoxia/reoxygenation. The concomitant attenuation of the production of IL-6 by astrocytes exposed to hypoxia/reoxygenation when RA301 expression was blocked by antisense oligonucleotide suggests that RA301 may affect synthesis of critical gene products.