(Received for publication, October 25, 1995; and in revised form, December 14, 1995)
From the
As the most abundant cell type in the central nervous system,
astrocytes are positioned to nurture and sustain neurons, especially in
response to cellular stresses, which occur in ischemic cerebrovascular
disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K.,
Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T.,
and Stern, D.(1996) J. Neurochem., in press), we identified
five polypeptide bands on SDS-polyacrylamide gel electrophoresis,
corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa,
whose expression was induced/enhanced in astrocytes exposed to hypoxia
or hypoxia followed by replacement into the ambient atmosphere
(reoxygenation). In the current study, the 150-kDa polypeptide has
been characterized. Chromatography of lysates from cultured rat
astrocytes on fast protein liquid chromatography Mono Q followed by
preparative SDS-polyacrylamide gel electrophoresis led to isolation of
a
150-kDa band only observed in hypoxic cells and which had a
unique N-terminal sequence of 15 amino acids. Antisera raised to either
the purified
150-kDa band in polyacrylamide gels or to a synthetic
peptide comprising the N-terminal sequence detected the same
polypeptide in extracts of cultured rat astrocytes exposed to hypoxia;
expression was not observed in normoxia but was induced by hypoxia
within 24 h, augmented further during early reoxygenation, and
thereafter decreased to the base line by 24 h in normoxia. ORP150
expression in hypoxic astrocytes resulted from de novo protein
synthesis, as shown by inhibition in the presence of cycloheximide. In
contrast to hypoxia-mediated induction of the
150-kDa polypeptide,
neither heat shock nor a range of other stimuli, including hydrogen
peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its
expression, suggesting selectivity for production of ORP150 in response
to oxygen deprivation, i.e. it was an oxygen-regulated protein
(ORP150). Northern and nuclear run-off analysis confirmed the apparent
selectivity for ORP150 mRNA induction in hypoxia. Subcellular
localization studies showed ORP150 to be present intracellularly within
endoplasmic reticulum and only in hypoxic astrocytes, not cultured
microglia, endothelial cells, or neurons subject to hypoxia. Consistent
with these in vitro results, induction of cerebral ischemia in
mice resulted in expression of ORP150 (the latter was not observed in
normoxic brain). These data suggest that astroglia respond to oxygen
deprivation by redirection of protein synthesis with the appearance of
a novel stress protein, ORP150. This polypeptide, selectively expressed
by astrocytes, may contribute to their adaptive response to ischemic
stress, thereby ultimately contributing to enhanced survival of
neurons.
Astrocytes are strategically located to exert neurotrophic
functions, especially in response to environmentally stressful
situations that threaten neuronal survival but are well tolerated by
astrocytes(2, 3) . Several examples of this beneficial
impact of astrocytes on neuronal homeostasis include their ability to
enhance the viability of neuronal cells subjected to glucose
deprivation (4) and hypoxia(5) . Mechanisms underlying
these effects are likely to be complex, and they include such factors
as regulation of extracellular potassium concentration (6) and
glutamate uptake(7) . Astrocytes can also express neurotrophic
factors(8) . In this context, we have reported that cultured
rat astrocytes exposed to hypoxia followed by reoxygenation elaborate a
neurotrophic cytokine, interleukin 6 (IL-6), ()suggesting
that this is an inducible factor produced by astrocytes that could
enhance neuronal survival(9) .
Deprivation of environmental
oxygen alters cellular biosynthetic and other properties, resulting in
a rapid shift to anaerobic glycolysis (1, 10) ,
depression of cellular proliferation(11) , and the production
of cytokines and growth
factors(12, 13, 14, 15) . Another
aspect of the cellular response to hypoxia is characterized by the
expression of a set of stress proteins, termed oxygen-regulated
proteins (ORP)(16) , which are believed to have important roles
in protecting cellular biosynthetic activities under stressful
circumstances. In cultured astrocytes subjected to hypoxia, expression
of a group of stress proteins, with molecular mass values corresponding
to 28, 33, 78, 94, and 150 kDa, was observed. The 28-, 78-, and
94-kDa stress proteins had properties of glucose-regulated proteins
(GRPs), based on parallel induction in normoxic astrocytes exposed to
2-deoxyglucose or calcium ionophore. Furthermore, the 78-kDa
polypeptide proved to be identical to GRP78/Bip by the N-terminal amino
acid sequence analysis(1) .
In our previous
study(1) , we observed an 150-kDa polypeptide expressed by
hypoxic astrocytes that has been characterized in detail in the current
experiments. The
150-kDa polypeptide is selectively expressed by
cultured rat astrocytes but not endothelial cells, microglia, or
neurons subjected to oxygen deprivation. Identification of the first 15
N-terminal amino acid residues indicates that this polypeptide is
unique. Based on its induction in hypoxic astrocytes but not in
astrocyte cultures exposed to heat shock, hydrogen peroxide,
2-deoxyglucose, or other agents, it has been termed an oxygen-regulated
protein-150 or ORP150. ORP150 is present intracellularly, localized to
the endoplasmic reticulum, and induced in gerbil brain following
experimentally induced ischemia. These data suggest that ORP150 may be
a selective marker of astrocyte adaptation to oxygen deprivation,
potentially enhancing the synthesis and/or export of proteins in the
endoplasmic reticulum important for astrocyte and, possibly, neuronal
survival in ischemia.
Cells were
plated at a density of about 5 10
cells/cm
in the above medium except neurons, which were cultured in
astrocyte-conditioned medium supplemented with fetal calf serum (10%)
and glucose (20 mM). When cultures achieved confluence, they
were exposed to hypoxia using an incubator attached to a hypoxia
chamber (Coy Laboratory Products, Ann Arbor, MI), which maintained a
humidified atmosphere with low oxygen tension, as described
previously(1, 19) . Where indicated, after exposure to
hypoxia, cultures were returned to the ambient atmosphere
(reoxygenation, R), at which time the conditioned medium was rapidly
exchanged with fresh medium. Oxygen tension in the medium was measured
using a blood gas analyzer (ABL-2, Radiometer, Sweden). Cell viability
was assessed by several methods including morphological criteria,
trypan blue exclusion, lactose dehydrogenase release, and evaluation of
general protein synthesis based on incorporation of
[
H]leucine into trichloroacetic acid-precipitable
material(20) .
To assess the specificity of anti-ORP150 IgG, Western blotting was performed by the method of Towbin et al.(23) . In brief, either normoxic or hypoxic astrocytes were exposed to Nonidet P-40 (1%), and about 10 µg of extracted protein (in each case) was subjected to SDS-PAGE (7.5%), transferred to PVDF paper, and reacted with anti-ORP150 IgG (1:200 dilution, 5 µg/ml) raised against the purified native ORP150. Fractions (2 µl) from FPLC Mono Q of hypoxic astrocyte extracts were similarly subjected to Western blotting using the anti-ORP150 IgG.
To further test the specificity of
anti-ORP150 IgG, studies were performed with synthetic peptide
comprising the first 15 amino acid residues of ORP150. The indicated
amount of either ORP150 N-terminal peptide or bovine serum albumin was
blotted onto nitrocellulose paper and reacted with anti-ORP150 IgG (5
µg/ml) raised to purified ORP150 as described above. In brief,
protein extract prepared from about 5 10
astrocytes
exposed to hypoxia for 48 h was incubated in Tris-buffered saline
(about 12 ml) containing Nonidet P-40 (1%), EDTA (5 mM), and
PMSF (1 mM) and was incubated for 12 h at 4 °C with either
anti-ORP150 IgG or preimmune IgG (1:50 dilution, 20 µg/ml in each
case). Then, a suspension of Staphylococcus aureus protein A
(0.4 ml/tube, 10% suspension of IgGSorb, The Enzyme Center, Malden, MA)
was added to each tube and incubated for 1 h at 4 °C. After
centrifugation (4000 rpm for 10 min), the supernatant was collected,
concentrated 50-fold by ultrafiltration, an aliquot (20 µl
containing about 10 µg of protein) was subjected to Western
blotting, and blots were reacted with anti-ORP150 IgG raised to the
N-terminal ORP150 synthetic peptide.
Immunocytochemical studies were performed in hypoxic astrocytes as described(25) . In brief, astrocytes plated on glass coverslips were exposed to hypoxia for 48 h, fixed in paraformaldehyde (3.3%), permeabilized using PBS containing Nonidet P-40 (1%), and incubated with either anti-ORP150 IgG (raised to purified ORP150) or preimmune rabbit IgG (in each case at 1:100 dilution, 10 µg/ml). Sites of primary antibody binding were visualized using rhodamine-conjugated anti-rabbit IgG (Sigma).
To further analyze the subcellular
localization of ORP150 antigen in hypoxic astrocytes, about 5
10
hypoxic astrocytes were pelleted and fractionated as
described(26) . In brief, astrocyte cell pellets frozen at
-80 °C were thawed, resuspended in 10 ml of buffer A (0.25 M sucrose, 10 mM Hepes-NaOH, pH 7.5, 1 mM dithiothreitol, 1 mM PMSF, 1 µg/ml leupeptin, 0.1
mM 1-chloro-3-tosylamido-7-amino-2-heptanone), and the cells
were cavitated at 400 psi N
pressure for 30 min by nitrogen
cavitation bomb (Kontes Glass Co., Vineland, NJ). Following
homogenization, the cell lysate was clarified by centrifugation at
10,000
g for 15 min at 4 °C, and the pellet was
resuspended in TNE buffer (10 mM Tris-HCl, pH 8.0, 1% Nonidet
P-40, 150 mM NaCl, 1 mM EDTA, 10 µg/ml aprotinin,
1 mM PMSF). The clarified lysate was then centrifuged and
fractionated by a series of sucrose steps: 38, 30, and 20% sucrose (all
prepared in 10 mM Hepes-NaOH, pH 7.5, 1 mM dithiothreitol) at 100,000
g for 3 h at 4 °C.
Layered fractions (fractions 1-4) were collected by puncturing
the tube at the desired depth, and gently withdrawing the fluid. The
pellet at the bottom of the tube was resuspended in 3 ml of buffer A
(precipitate fraction). Following measurement of protein concentration,
each fraction (about 1 µg of protein) was subjected to Western
blotting using either anti-ORP150 IgG raised to purified ORP150 or
anti-GRP78 antiserum.
Nuclear run-off analysis was performed as described (9) to
determine the relative rates of ORP150 transcription. In brief, nuclear
suspension (0.2 ml), obtained from about 2 10
astrocytes prepared under the indicated conditions, was incubated
with CTP, ATP, and GTP (0.5 mM each) in the presence of
[
-
P]UTP (250 µCi, 3000 Ci/mmol; DuPont
NEN). Samples were subjected to phenol/chloroform extraction, and RNA
was precipitated and resuspended in hybridization buffer. Hybridization
to denatured rat ORP150 and rat
-actin (control) probes (20 µg
in each case) dot-blotted onto nylon membranes was performed at 42
°C for 2 days. Filters were washed, dried, and exposed to Fuji
x-ray film.
Figure 1:
Isolation of ORP150
from rat cultured astrocytes by FPLC Mono Q (A and B)
and preparative SDS-PAGE (C). A, protein extracts
from astrocytes (about 5 10
cells, 2-3 mg of
protein) exposed to hypoxia for 48 h were filtered, diluted with 50 ml
of PBS, and applied to FPLC Mono Q. The column was washed with 0.2 M NaCl and eluted with an ascending salt gradient; protein
content in eluate was monitored by A
. B, reduced SDS-PAGE (7.5%) was performed on protein extracts
from either normoxic or hypoxic astrocytes (about 10 µg of protein
each, lanes N and H) and on aliquots of fractions (10
µl, fractions 1-20) from the Mono Q column (0.5 ml, lanes 1-20). Proteins in the gel were visualized by
silver staining, and migration of simultaneously run molecular weight
markers is indicated on the far left side of the gel
(ovalbumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (97.4 kDa),
-galactosidase (116 kDa), and myosin (200 kDa)).
The migration of ORP150 is indicated by the asterisk. C, fractions eluted from FPLC Mono Q, which contained the
150-kDa polypeptide (fractions 6 and 7) were
pooled and concentrated by ultrafiltration. About 20 µg of protein
was applied to reduced SDS-PAGE, and protein was detected by staining
with Coomassie Blue. The asterisk indicates the migration of
ORP150, and migration of molecular weight markers is shown on the far right side of the gel.
Figure 2:
Immunoblotting of ORP150 using antibody
raised against purified ORP150 or the N-terminal synthetic peptide. A, immunoblotting using anti-ORP150 antibody raised against
purified ORP150. Extract from either normoxic(N) or hypoxic (H)
astrocytes (about 10 µg in each case) or aliquots of fractions from
FPLC Mono Q (1-20, from Fig. 1A) were
subjected to reduced SDS-PAGE (7.5%) and transferred to PVDF paper.
After blocking excess sites on the paper, PVDF was reacted with
anti-ORP IgG (5 µg/ml), and sites of primary antibody binding were
visualized as described in the text. B, the indicated amount
of ORP150 synthetic peptide (ORP150) or bovine serum albumin (BSA) were
dot-blotted on nitrocellulose paper and reacted with rabbit IgG
obtained either before (Preimmune) or after (Postimmune) immunization
of animals with purified ORP150. Sites of primary antibody binding were
visualized as above. C, lysate of hypoxic astrocytes (from
about 5 10
cells) was pre-adsorbed with either
anti-ORP150 IgG raised against purified ORP150 (
ORP150) or
preimmune IgG (preimmune) as described in text. After the removal of
IgG-ORP150 complexes using immobilized protein A, supernatant was
concentrated (to about 20 µl in each case) and subjected to reduced
SDS-PAGE (7.5%). Other samples applied to the gel were lysate of
hypoxic (HYPOXIA) or normoxic (NORMOXIA) astrocytes (about 1 µg of
protein). Following Western blotting, membranes were reacted with
anti-ORP150 IgG (5 µg/ml) raised against the N-terminal synthetic
peptide (sites of primary antibody binding were detected as described
in the text). In panels A and C, migration of
simultaneously run molecular weight markers is shown on the left.
Figure 3:
Induction of ORP150 in cultured astrocytes
by hypoxia and hypoxia/reoxygenation. Cultured astrocytes (about 5
10
cells) were exposed to either hypoxia alone
(HYPOXIA, 0-48 h) or hypoxia followed by reoxygenation (Reox,
2-24 h). At the indicated times, cells were washed and lysed in
the presence of PBS containing Nonidet P-40; after the centrifugation
to remove debris, the supernatant (about 1 µg of protein) was
applied to SDS-PAGE (7.5%). Protein in the gel was transferred to PVDF
paper and incubated with anti-ORP150 IgG (ORP150). The migration of
simultaneously run molecular weight markers is shown on the far
right. The same samples were also immunoblotted with anti-GRP78
antiserum (GRP78), anti-HSP72 antibody (HSP72), or anti-HO-1 antiserum
(HO-1), and the relevant portion of the blot is
shown.
To further characterize astrocyte
induction of ORP150, its expression in astrocyte lysates exposed to
chemical agents was studied by immunoblotting. Treatment of normoxic
astrocyte cultures with either 2-deoxyglucose or tunicamycin resulted
in the expression of GRP78 (Fig. 4, GRP78). HO-1 antigen was
induced in astrocytes by addition of hydrogen peroxide, cobalt
chloride, or elevated temperature (Fig. 4, HO-1); whereas only
elevated temperature induced HSP72 (Fig. 4, HSP72). None of
these chemical stimuli elicited expression of ORP150 antigen in
astrocytes (Fig. 4, ORP150). The role of de novo protein biosynthesis in ORP150 expression by hypoxic astrocytes
was emphasized by disappearance of the ORP150 band from cultures
subjected to hypoxia in the presence of cycloheximide (3 µg/ml);
this concentration of cycloheximide reduced overall protein synthesis
by about 85%, based on incorporation of
[H]leucine into material precipitable in
trichloroacetic acid (Fig. 4, ORP150, Hypoxia and Hypoxia +
Cx (H+CX), respectively). These data indicated the specificity of
ORP150 expression for oxygen deprivation as the result of redirection
of cellular protein synthesis.
Figure 4:
Induction of ORP150 in cultured astrocytes
by chemical agents or elevated temperature. Cultured astrocytes (about
5 10
cells) were exposed to either normoxia (NORMO)
or hypoxia for 24 h in the absence (HYPO) or presence of cycloheximide
(3 µg/ml, H+CX). Astrocytes maintained in normoxia were
subjected to either heat shock (43 °C for 3 h, HEAT); exposure to
hydrogen peroxide (5 µM for 10 min, H
O2),
followed by the incubation for 6 h; or exposure to cobalt chloride (1
mM, Co), 2-deoxyglucose (25 mM, 2DG), or tunicamycin
(5 µg/ml, TM) for 24 h. Cells were then harvested as in Fig. 3, and about 1 µg from each sample was subjected to
immunoblotting using the same antibody
preparations.
Figure 5:
Cellular localization of ORP150. A, subcellular fractionation of hypoxic astrocyte lysates.
About 10 astrocytes were exposed to hypoxia for 48 h,
harvested by centrifugation, and fractionated as described as text.
Each fraction (about 1 µg of protein) was subjected to
immunoblotting using anti-ORP150 IgG prepared against purified ORP150.
Each lane shows a fraction containing washed membrane pellet (lane
1), membrane wash (lane 2), membranes (lane 3),
cytosolic protein (lane 4), cytosolic protein and membranes (lane 5), nuclear wash (lane 6), purified nuclear
extract (lane 7), nuclear debris (lane 8), purified
nuclei (lane 9), or crude nuclear pellet (lane 10). B, immunocytochemical analysis of hypoxic astrocytes.
Astrocytes plated on coverslips were exposed to hypoxia for 48 h,
permeabilized, and incubated with anti-ORP150 IgG (B1),
anti-GRP78 antiserum (B2), or preimmune IgG (B3).
Sites of primary antibody binding were detected using
rhodamine-conjugated anti-rabbit IgG. Magnification, 400
. C, sucrose density gradient centrifugation of hypoxic
astrocyte lysates. Cavitants of hypoxic astrocytes (about 5
10
cells) were further separated by sucrose gradient as
described in the text. Each fraction (about 1 µg of protein) was
immunoblotted using anti-ORP150 IgG (ORP150) or anti-GRP78 antiserum
(GRP78). Lanes represent fractions containing cytosol (1),
plasma membrane (2), Golgi (3), and endoplasmic
reticulum (4 and 5). Migration of simultaneously run
molecular weight markers is shown on the right for the blot
with anti-ORP150 IgG, and the relevant portion of the blot with
anti-GRP78 antiserum is shown.
Figure 6:
Induction of ORP150 mRNA in cultured
astrocytes by hypoxia or hypoxia/reoxygenation. Astrocytes (about 2
10
cells) were exposed to hypoxia (HYPOXIA; 0, 24,
and 48 h) or hypoxia followed by reoxygenation (REOX; 2, 4, 12, and 24
h). At the indicated time point, total RNA was extracted, subjected to
agarose/formamide electrophoresis, followed by transfer onto Biodyne B
filters. Filters were hybridized overnight at 42 °C in
hybridization buffer containing either radiolabeled ORP150 probe
(ORP150) or radiolabeled
-actin probe (
-actin). Filters were
then washed, dried, and subjected to autoradiography. Migration of 18
and 28 S ribosomal RNA is indicated on the left.
Figure 7:
Effect of hypoxia and
hypoxia/reoxygenation on the transcription of ORP150 mRNA. Nuclei were
prepared from about 2 10
astrocytes exposed to
hypoxia (H 24 and H 48) or hypoxia followed by reoxygenation for 2 or 8
h (H/R 2 and H/R 8), incubated with CTP, ATP, and GTP in the presence
of [
-
P]UTP; RNA was extracted and
hybridized to denatured rat ORP150 or rat
-actin (control) probes
(20 µg) dot-blotted onto nylon membranes. Filters were then washed,
dried, and subjected to autoradiography (H0 represents normoxic
control).
Figure 8:
Expression of ORP150 in other cell types
of the central nervous system. Astrocytes (ASTRO), microglia (MCG),
brain microvessel endothelial cells (BMVEC), and cortical neurons
(NEURON) were separated, cultured, and exposed to either hypoxia (H) or
normoxia(N) for 24 h (about 5 10
cells in each
case). Cells were lysed, and about 1 µg of protein extract was
subjected to immunoblotting using the anti-ORP150 IgG prepared to the
purified protein as described. Migration of molecular weight markers is
shown on the left.
Figure 9:
Expression of ORP150 antigen in ischemic
brain. A, immunoblotting. Adult C57DL1 mice (20-25 g)
were subjected to permanent occlusion of the middle cerebral artery for
24 h. The cortex area of either ischemic side (Ischemic) or
non-ischemic side (Control) was harvested, homogenized in PBS
containing Nonidet P-40, and subjected to immunoblotting using
anti-ORP150 IgG raised to purified ORP150. Migration of molecular
weight markers is shown on the left. B,
immunohistochemistry demonstrates expression of ORP150 antigen
(visualized with anti-ORP150 IgG, 5 µg/ml) in ischemic side of the
mouse brain (right side) versus its absence in
nonischemic control side (left side). Magnification,
3.5.
Analysis of the astrocyte response to environmental challenge is particularly important as these cells withstand stressful conditions that curtail neuronal viability(2, 3) . In addition, astrocytes can sustain neuronal homeostasis through a variety of mechanisms, including the elaboration of neurotrophic factors. In a previous study, we found that cultured astrocytes exposed to hypoxia followed by replacement into normoxia (H/R) elaborated the neurotrophic cytokine IL-6, which enhanced survival of PC12(9) . To analyze mechanisms underlying the synthesis and release of IL-6 by astrocytes exposed to H/R, we characterized new products of protein synthesis under these conditions. Of the five polypeptides whose expression was dramatically enhanced/induced by hypoxia or H/R, based on SDS-PAGE analysis, we first characterized a 78-kDa polypeptide, which proved to be identical to GRP78 by N-terminal sequencing and reactivity with anti-GRP78 antisera(1) . GRP78 functioned as a chaperon to enhance the elaboration of IL-6 by astrocytes exposed to H/R; in fact, IL-6 was present in conditioned media complexed with GRP78, and addition of antisense oligonucleotides for GRP78 suppressed release of IL-6. These data suggested the complexity of events activated by hypoxia and H/R for biosynthetic mechanisms likely to be affecting pathways from transcription to protein processing and release from the cell.
In this report, we have characterized a 150-kDa
polypeptide whose expression was induced by hypoxia or H/R in
astrocytes. N-terminal sequence analysis defined this polypeptide as
being unique, which is consistent with our preliminary analysis of the
recently isolated partial cDNA (corresponding to deduced amino acid
residues 1-884).
Furthermore, the
150-kDa
polypeptide is produced selectively in response to oxygen deprivation,
leading us to assign the name ORP150. This contrasts with HO-1 and
GRP78, both of which display enhanced expression in response to
hypoxia, as well as other stimuli. Our data suggest it is unlikely that
heme-containing proteins, implicated in the induction of heme oxygenase
or erythropoietin by heavy metals such as
cobalt(30, 31) , are involved in ORP150 expression. In
addition, the lack of ORP150 induction following inhibition of
glycolysis, following addition of 2-deoxyglucose, which induces GRP78 (1) and other GRPs(32) , also points to the likelihood
that distinct mechanisms underly ORP150 expression, possibly mechanisms
more directly related to oxygen deprivation.
Another facet of ORP150 induction deserving of comment concerns its selective expression in astrocytes, as opposed to other cell types, exposed to hypoxia or H/R. In cell culture, neurons, microglia, nor endothelial cells subjected to similar environmental conditions expressed ORP150. By contrast, polypeptides such as HO-1 and GRP78 are expressed in diverse cell types. Furthermore, in the mouse model of ischemia injury, immunocytochemical studies showed expression of ORP150 in ischemic area, suggesting that induction of ORP150 is part of the central nervous response to oxygen deprivation. These data suggest that ORP150 may be a marker of ischemic stress and lead us to speculate that it may have an important role in the successful adaptation of astrocytes to oxygen deprivation. In this context, the presence of ORP150 in endoplasmic reticulum places it at the critical locus for controlling de novo protein synthesis and processing.
These studies represent a first step in the characterization of a novel polypeptide induced in astrocytes by hypoxia or H/R. While the functional role of ORP150 to the adaptive astrocyte response to ischemic stress, including expression of neurotrophic stimuli, remains to be defined, we have developed the reagents and tools that will permit this next level of analysis.