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INTRODUCTION |
Cellular adaptations to environmental oxygen deprivation
constitute an important protective pathway for the host response to
ischemia (1). One essential component of this response includes increased dependence on anaerobic metabolism as an energy source (2,
3). Enhanced glycolysis is facilitated by hypoxia-mediated up-regulation of the noninsulin-dependent glucose
transporter-1 and several enzymes involved in glycolytic metabolism by
a mechanism that involves hypoxia-inducible factor-1. Another facet of
the cellular biosynthetic response to hypoxia involves expression of
diverse oxygen-regulated proteins
(ORPs)1 (4). These stress
proteins participate in many aspects of cellular functions, and their
characterization provides important clues as to means through which
cells sustain hypoxic injury.
Among cell populations in the central nervous system, astrocytes are
programmed to withstand hypoxic and ischemic stress, compared with more
vulnerable neurons (5). Because of their strategic location, astrocytes
are thought to have neurotrophic roles in response to cell stress, as
observed in inflammation (6), trauma (7), and ischemic cerebrovascular
diseases (8). For this reason, we have characterized proteins whose
expression is modulated in astrocytes subjected to oxygen deprivation.
We have previously identified several stress proteins in hypoxic cultured astrocytes; polypeptides with molecular masses corresponding to 94, 78, 33, and 28 kDa proved to be identical to GRP94, GRP78/Bip, heme oxygenase-1, and HSP28, respectively (2). A major
hypoxia-inducible stress protein with a mass of 150 kDa was cloned and
characterized as ORP150 (9, 10), a new member of heat shock protein
family located in the endoplasmic reticulum. ORP150 is likely identical to a previously described polypeptide with a mass of 170 kDa, observed
as a band with the latter mobility on SDS-polyacrylamide gel
electrophoresis in hypoxic Chinese hamster ovary cells (11). The
localization of ORP150, GRP94, and GRP78/Bip (12) to the endoplasmic
reticulum suggests that hypoxia induces a stress response whose focal
point may be in this organelle.
Because of the striking induction of ORP150 expression in cultured
astrocytes (10) and mononuclear phagocytes (13) exposed to hypoxia, we
speculated that this protein might have a cytoprotective role in
response to cell stress associated with oxygen deprivation. To address
this issue, we have established a cell line stably transfected to
overexpress ORP150 antisense RNA in human embryonic kidney (HEK) cells.
Antisense-transfected HEK cells display increased vulnerability to
hypoxia, with loss of cell viability due to apoptosis, compared with
wild-type and sense-transfected cultures. These data indicate that
ORP150 has a pivotal role in cytoprotective cellular mechanisms
triggered by oxygen deprivation.
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MATERIALS AND METHODS |
Construction of the ORP150 Antisense Vector--
The vector
pCAGGS, provided by Dr. Jun-ichi Miyazaki (14), contains a neomycin
resistance gene linked to the thymidine kinase promoter. To construct
the ORP150 antisense/sense vector, a fragment encoding almost the
entire human ORP150 cDNA, as well as some 3'-untranslated sequence
(residues 86-3093) (9), was inserted into the pCAGGS vector in either
antisense or sense orientation. This was accomplished with amplicons
prepared using primers with EcoRI linkers
5'-cgaattcctcGAGGCAAGAGGGGCACTATG-3' and
5'-gggaattctcGAGGTGGGGGTTATA-GTTCG-3' (EcoRI linker underlined). After sequencing of
polymerase chain reaction products, the orientation of the inserted
ORP150 cDNA fragment was confirmed by both restriction enzyme
mapping and DNA sequencing.
Introduction of pCAGGS-Antisense/Sense ORP150 into HEK Cells and
Selection of Stable Transfectants--
Ten micrograms of vectors, with
or without ORP150-antisense/sense, were transfected into HEK cells
using the Tfx (Promega) Lipofectamine method. Selection and maintenance
of the neo-resistant transfectants were performed in the presence of
G418 (Sigma, 1.5 mg/ml). After 14 days, single colonies were
resuspended and grown in 96-well plates at a density of about one cell
per well. Several cell lines were isolated, all of which are maintained
in the presence of G418 (1.5 mg/ml). Cells were switched to the
G418-free medium 24 h prior to experiments.
Induction of Hypoxia--
Cells were plated at a density of
about 5 × 104 cells/cm2 in Dulbecco's
modified Eagle's medium containing fetal calf serum (10%) and
penicillin/streptomycin (100 units/ml, 100 µg/ml). Where indicated,
HEK transfected with either antisense, sense, or vector-only construct
were maintained in media containing G418 (800 µg/ml). Prior to
experiments (24 h), culture medium was changed to the same medium
without G418 for both wild-type and sense/antisense-transfected HEK
cells. When cultures achieved confluence (which took the same time for
wild-type HEK cells, sense, antisense, or vector-only HEK
transfectants), they were exposed to hypoxia using an incubator attached to an hypoxia chamber (Coy Laboratory Products, Ann Arbor, MI)
which maintained a humidified atmosphere with low oxygen tension (8-10
torr), as described previously (15). Oxygen tension in the medium was
measured using a blood gas analyzer (ABL-2, Radiometer, Sweden).
Western Blot and Northern Blot Analyses--
Cultured cells
(about 5 × 106 cells) were exposed to hypoxia, and,
at the indicated time points, cells were washed three times with
ice-cold phosphate-buffered saline (PBS) and lysed in the presence of
PBS (200 µl) containing Nonidet P-40 (1%), EDTA (5 mM),
and phenylmethylsulfonyl fluoride (1 mM). After
centrifugation (5000 × g for 5 min at 4 °C),
protein concentration of each supernatant was determined by the Lowry
method (16). Samples were applied to SDS-polyacrylamide gel
electrophoresis (7.5%), transferred to polyvinylidene difluoride
membranes, and visualized with polyclonal anti-human ORP150 IgG (0.15 mg/ml; purified by protein A column) raised to recombinant ORP150
polypeptide (residues 508-999) (17). Where indicated, samples were
also subjected to immunoblotting with anti-KDEL monoclonal antibody
(Stressgen, Canada) according to the manufacturer's protocol.
Expression of ORP150 mRNA in HEK cells exposed to hypoxia employed
a partial ORP150 cDNA corresponding to 151-570 base pairs (amino
acids 51-190) for Northern analysis, as described (13). About 20 µg
of total RNA was extracted from HEK cells exposed to hypoxia or
normoxia for the indicated times by the AGPC method; RNA was separated
by electrophoresis on 1.0% agarose/formamide gels and transferred
overnight onto Biodyne B paper (Pall BioSupport, New York). The
membrane was prehybridized for 3 h at 42 °C in hybridization
buffer (0.9 M NaCl, 90 mM sodium citrate, pH
7.0) containing 5× Denhardt's solution, SDS (0.5%), and
heat-denatured salmon sperm DNA (100 µg/ml). ORP150 cDNA was
radiolabeled with [32P]dCTP (NZ522, NEN Life Science
Products) by the random hexamer procedure (18). After hybridization
overnight at 42 °C in hybridization buffer containing radiolabeled
cDNA probe (5 ng/ml), filters were washed twice with 2× SSC, 0.5%
SDS and 0.2× SSC, 0.5% SDS for 30 min at 52 °C, exposed to x-ray
film (Fuji Photo Film, Japan), and subjected to autoradiography. Blots
were also hybridized with a radiolabeled probe for
glyceraldehyde-3-phosphate dehydrogenase to serve as a control for RNA loading.
Analysis of Cell Viability--
To assess cell viability,
wild-type HEK cells and stable transformant HEK cells transfected with
either antisense, sense, or vector-only construct were either exposed
to hypoxia or to sodium arsenate or hydrogen peroxide under normoxic
conditions. Release of lactate dehydrogenase (LDH) activity into the
culture medium was measured according to the manufacturer's protocol
(Cacatua Chemical Co., Tokyo, Japan). Levels of cytoplasmic
histone-associated DNA fragments were assayed using the Cell Death
enzyme-linked immunosorbent assay (Boehringer Mannheim, Mannheim,
Germany) according to the manufacturer's manual. In brief, the
cytosolic fraction (13,000 × g supernatant) of
approximately 500 cultured cells was used as antigen source in a
sandwich enzyme-linked immunosorbent assay with primary anti-histone
antibody coated to the microtiter plate and secondary anti-DNA antibody
coupled to peroxidase. From the absorbance values, the fold increase of
fragmentation was calculated according to Equation 1 (19) using either
normoxic or non-treated counterparts as control cells.
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(Eq. 1)
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Where indicated, HEK cells were placed in hypoxia (as above), or
in normoxia, in the presence of sodium arsenate (10 mM), or
hydrogen peroxide (10 mM for 30 min) and subsequently
incubated for 6 h prior to harvest. Cell proliferation was
determined by seeding 2.5 × 104 cells into
3.0-cm2 well and beginning the incubation period at
37 °C. At later time points, cells were collected by trypsinization,
and cell number was determined with a Coulter counter as described
(20). Mitochondrial function was also assessed by HEK cell
incorporation of dimethylthiazol-diphenyltetrazolium (Wako Chemicals)
as described (21). For this assay, cells were incubated for 3 h
with dimethylthiazol-diphenyltetrazolium (500 µg/ml), lysed with HCl
(0.05 M)/isopropyl alcohol, and
A570 nm was determined.
DNA Fragmentation--
In addition to the enzyme-linked
immunosorbent assay for cytoplasmic histone-associated DNA fragments,
DNA fragmentation was directly evaluated by agarose gel electrophoresis
(22). For isolation of DNA, about 105 of HEK cells were
resuspended in 200 µl of ice-cold PBS, containing 0.5 mg/ml
proteinase K, 0.5 mg/ml RNase A, and 1% SDS, and incubated at 37 °C
for 30 min. After addition of 300 µl of NaI solution (6 M
NaI, 13 mM EDTA, 0.5% sodium
N-lauroylsarcosine, 10 mg/ml glycogen, and 26 mM
Tris-HCl, pH 8.0), tubes were incubated at 60 °C for 15 min. DNA in
the supernatant (fragmented DNA) was precipitated in isopropyl alcohol,
air-dried, and analyzed on 2% agarose gels by ethidium bromide
staining and ultraviolet transillumination.
Morphologic and Ultrastructural Analysis--
Fluorescence
microscopy utilized ORP150 antisense transfectants (106
cells) cultured under hypoxia for 18 h. Cells were washed twice with ice-cold PBS, stained with propidium iodide (5 µg/ml), and analyzed by fluorescence microscopy with excitation at 360 nm. Quantitative assessment of propidium iodide-positive cells was performed by counting the number of fluorescent cells by an
investigator without knowledge of the experimental protocol. Transfer
of phosphatidylserine to the outer leaflet of the plasma membrane was
quantitatively assessed by increased binding of annexin V (23) using a
kit (Genzyme) according to the manufacturer's instructions. In brief, either wild-type or antisense-transfected HEK cells were exposed to
hypoxia for the indicated periods, washed three times in PBS, and
incubated with fluorescein isothiocyanate-labeled annexin V for 20 min
at 37 °C. After extensive washing in ice-cold PBS, cells were
examined by fluorescence microscopy with excitation at 488 nm, and
positive cells were quantitated by an individual unaware of the
experimental protocol.
Electron microscopic analysis utilized either wild-type or ORP150
antisense transfectants exposed to hypoxia for the indicated periods
and fixed in 6-well culture dishes in cold picric acid (0.2%),
paraformaldehyde (4%), and glutaraldehyde (0.05%) in 0.1 M phosphate buffer, pH 7.4, for 2 h at 4 °C. Cells
were washed in phosphate buffer, post-fixed for 1 h with 1%
osmium tetroxide (OsO4) in 0.1 M phosphate
buffer, pH 7.4, at 4 °C, and dehydrated. Ultrathin sections were
stained with lead citrate and subjected to ultrastructural analysis.
Measurement of Adenosine Nucleotide--
The content of high
energy adenosine metabolites was measured as described previously (3).
In brief, either antisense transfectants or wild-type HEK cells (about
106 cells) plated on 9.6-cm2 wells were exposed
to hypoxia. At the indicated times, cultures were washed with ice-cold
PBS, pelleted, and lysed in 400 µl of ice-cold perchloric acid (1 M). After centrifugation (4,000 × g for 5 min), the supernatant was neutralized with KOH (2 M) and subjected to reversed phase high pressure liquid chromatography (C18 column, Rainin). Pellets were stored for assay of
protein content. The peak absorbance at 260 nm corresponded to ATP and was identified by its retention time on the column. The content of each
adenosine metabolite (ATP, ADP, and AMP) was calculated using
Chromatopack (Shimazu, Kyoto, Japan).
Measurement of Caspase-1- and -3-like Activity and Western
Blotting for Cytosolic Cytochrome c and Caspase
Substrates--
Activity of Caspase-1- and -3-like protease was
assessed as described (22). In brief, either wild-type or ORP150
antisense-transfected HEK cells were exposed either to staurosporine (1 µM) in normoxia or to hypoxia for the indicated times. At
each time point, caspase-1- and -3-like activities were measured
colorimetrically by cleavage of 7-amino-4-methylcoumarin-YVAD and
7-amino-4-methylcoumarin-DEVD, respectively. One unit was defined as
the amount of enzyme required to release 0.22 nmol of AMC per min at
37 °C. Immunoblotting of cytoplasmic cytochrome c was
assessed by the Western blot as described (24).
To assess the possible contribution of caspase activation in the
hypoxia-mediated cell death in antisense-transformant cells, polyclonal
antibody for recombinant human caspase-9 was raised in rabbits. In
brief, full-length caspase-9 cDNA was fused into glutathione
S-transferase gene in plasmid pGEX-1T (Pharmacia Biotech, Uppsala, Sweden). Glutathione S-transferase-caspase-9
recombinant protein was expressed in Escherichia coli and
purified by affinity chromatography to immunize rabbits.
Western blot analysis for caspases and their substrates was then
performed using antibody raised to poly(ADP-ribosome) polymerase (25),
caspase-2 (26), caspase-7 (27; these reagents were purchased from Santa
Cruz Biotechnology, Santa Cruz, CA), caspase-8 (28; purchased from
Transduction Laboratories), and caspase-9 as described (29). In brief,
antisense-transfected HEK cells were either exposed to hypoxia or
staurosporine (1 µM) for the indicated time. Then cells
were pelleted, lysed in PBS containing Nonidet P-40 (1%), SDS (0.1%),
and deoxycholic acid (0.1%), and lysates were subjected to the Western blotting.
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RESULTS |
Characterization of Stable Transfectants Overexpressing
Antisense/Sense ORP150 Transcripts--
The ORP150 antisense/sense
construct was prepared by inserting a construct comprised of virtually
the full coding sequence of human ORP150 and a portion of
3'-untranslated sequence (+86/+3093) into the EcoRI site of
pCAGGS vector in either reverse or forward orientation (pCAGGS-ORP150
antisense or pCAGGS-ORP-150 sense, respectively; Fig.
1A). After transfection of
pCAGGS-ORP150 antisense/sense into HEK cells, transfectants were
selected and cloned in medium containing G418 and were designated
ORP150 antisense or sense transfectants. Compared with wild-type HEK
cells (Fig. 1B, lane 1) or cultures exposed to vector alone
(Fig. 1B, lane 2), immunoblotting of lysates from two clones
of ORP150 sense transfectants showed higher ORP150 antigen (Fig.
1B, lanes 3 and 4), whereas two clones of ORP150
antisense transformants displayed lower levels of ORP150 (Fig.
1B, lanes 5 and 6). Laser densitometry
indicated a 10-fold increase in ORP150 expression in sense
transfectants and about 40-fold suppression in antisense transfectants.
For the experiments shown below, studies were performed in parallel
with each of these cell lines, and representative results are
shown.

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Fig. 1.
Schematic representation of construction of
the sense/antisense ORP150 mRNA expression vector (pCAGGS-ORP150;
A), base-line expression of ORP150 in transfectants
(B), and the growth properties of wild-type, sense,
and antisense transfectants HEK cells under normoxia
(C). ORP150 fragment generated by polymerase
chain reaction amplification of +86/+3093 from plasmid pSPORT1-ORP150
was inserted into the EcoRI site of pCAGGS by using an
EcoRI linker oriented in either forward (sense)
or opposite (antisense) polarity to the SV40 promoter. The
arrows indicate the polarity of the genes (A).
B, detergent extract (1% Nonidet P-40; 10 µg/lane) from
either wild-type (lane 1) or stably transfected HEK cells
after transfection with either vector-only (lane 2), sense
vector (lanes 3 and 4), or antisense vector
(lanes 5 and 6) were cloned, and ORP150
expression was assessed by the Western blot analysis. The migration of
simultaneously run molecular mass markers is indicated on the
left side of the gel (ovalbumin (45 kDa), bovine serum
albumin (66 kDa), phosphorylase b (96 kDa),
-galactosidase (116 kDa), and myosin (200 kDa)). C,
wild-type HEK cells or ORP150 sense/antisense/vector-only transfectants
were incubated at an initial density of 2.5 × 104
cells per well (3 cm2) in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum and antibiotics in the absence
(wild-type HEK) or in the presence (antisense and sense transfectants)
of G418 for the indicated times. At the indicated time points, the
number of ORP150 antisense transfectants (open bar),
wild-type HEK cells (closed bar), ORP150 sense-transfectants
(hatched bar), or vector-only transfectants (dotted
bar) was assessed. Mean ± S.D. is shown (n = 12).
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Growth and Viability of ORP150 Sense/Antisense
Transfectants--
Proliferation of HEK cells, either ORP150
antisense/sense/vector-only transfectants compared with wild-type
cultures, was assessed by determining cell number (Fig. 1C)
or by the dimethylthiazol-diphenyltetrazolium method. In each case,
growth in cell culture over 5 days was comparable; there was no
significant difference among wild-type, antisense, and sense
transfectants to achieve confluence (data not shown), and no evidence
of increased cell death was noted by uptake of trypan blue (data not shown).
Expression of ORP150 in HEK Cell and Effect of Hypoxia on Cell
Viability in ORP150 Transfectants--
ORP150 was first identified by
its enhanced expression in hypoxic cultured astrocytes (10). Wild-type
HEK cells subjected to oxygen deprivation displayed a
time-dependent increase in ORP150 antigen first noted at
6-12 h and sustained up to the longest time point, 24 h (Fig.
2A, wild-type). In contrast,
levels of ORP150 in the antisense HEK transfectants increased only
slightly at 6-15 h (Fig. 2A, antisense). Consistent with
suppression of ORP150 expression in the antisense transfectants, ORP150
transcripts were virtually undetectable in ORP150 antisense
transfectants subjected to normoxia or hypoxia, compared with strong
up-regulation of ORP150 mRNA in wild-type HEK cultures (Fig.
2B). Although expression of ORP150 was suppressed in
antisense transfectants, hypoxia-mediated increase in the levels of two
glucose-regulated proteins, GRP78 and GRP94 (30), was similar in
wild-type (Fig. 2C, wild-type) and ORP150 antisense (Fig.
2C, antisense) transfectants. Thus, ORP150 antisense
transfectants showed a selective change in ORP150 antigen which did not
extend to other genes previously shown to undergo increased expression
in hypoxia.

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Fig. 2.
Expression of ORP150 antigen
(A), ORP150 transcripts (B), and
GRP78 and GRP94 antigens (C) in cultured wild-type HEK
cells and ORP150 antisense-transfected HEK cells exposed to
hypoxia. A, protein extract from either wild-type HEK
cells (20 µg/lane; left panel) or ORP150 antisense
transfectants (5 µg/lane; right panel) exposed to hypoxia
for the indicated times (0 means 0 h of hypoxia, as in the
normoxic control) was subjected to Western blotting as described in the
text. Note that a different amount of protein was loaded for the
antisense transfectant. B, same amount of protein (20 µg/lane) was loaded and subjected to Western blot using anti-KDEL
monoclonal antibody (Stressgen) for GRP78 and GRP94 detection.
C, total RNA (20 µg/lane) prepared from either wild-type
(wild) or antisense (anti)-transfected HEK cells
was subjected to Northern analysis with radiolabeled human ORP150
cDNA probe. The migration of ribosomal RNA is indicated on the
far right of the gel. GPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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In view of the association of increased ORP150 expression with
environmental hypoxia, we assessed whether ORP150 contributed to the
cellular adaptive response triggered by oxygen deprivation. ORP150
antisense transfectants displayed increased levels of cytoplasmic histone-associated DNA fragments within 12 h of hypoxia, which reached maximum within 18 h (Fig.
3A). Evidence of DNA
fragmentation was followed by release of LDH activity in the culture
supernatant (Fig. 3B). In contrast, both wild-type HEK cells
and sense transfectants did not show loss of viability up to 30 h.
Similar results were observed with vector alone HEK transfectants (Fig.
3, A and B).

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Fig. 3.
Viability of wild-type HEK cells and ORP150
antisense-transfected HEK cells in hypoxia. Either
antisense-transfected HEK cells (open bar), wild-type HEK
cells (closed bar), sense-transfected HEK cells
(hatched bar), or vector-only transfectants (dotted
bar) were exposed to hypoxia up to 30 h. At the indicated
times, fragmented DNA (A) and LDH activity (B) in
the culture supernatant was measured as described in text. Note that
all values of each group at time "0" in A are equal to
1.0 according to the formula described in text. Mean ± S.D. is
shown (n = 6) for DNA fragmentation. Mean ± S.D.
is shown (n = 24) for LDH release.
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Since ORP150 expression was associated with protection from
hypoxia-induced cell death, the effect of other stimuli on ORP150 antisense transfectants was evaluated. Cell viability was
comparable in wild-type and ORP150 antisense transfectants following
incubation of cultures with sodium arsenate, a potent degrading agent
for cellular protein, and hydrogen peroxide, a generator of oxygen free
radicals (Fig. 4, A and
B).

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Fig. 4.
Cell viability of Wild HEK cells and ORP150
antisense transformant HEK cells under chemical stresses (A
and B). Either wild-type HEK cells
(closed bar) or ORP150 antisense-transfectants HEK cells
(open bar) were cultured in the presence of either sodium
arsenate (10 mM; A) or hydrogen peroxide (10 mM, B) up to 24 h. At the indicated times,
LDH activity in each culture supernatant was measured, and cell
viability was expressed as a percentage of maximal release. Mean ± S.D. is shown (n = 24). In each case, experiments
were repeated a minimum of four times.
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Hypoxia-induced Cell Death in ORP150 Antisense Transfectants:
Evidence of Apoptosis--
Several lines of evidence demonstrated that
accelerated cell death in ORP150 antisense transfectants was due to
apoptosis. First, addition of cycloheximide suppressed cell death in
ORP150 antisense transfectants (Fig. 5,
A and B). In the presence of cycloheximide (2 µ/ml), overall protein synthesis was suppressed by >90%, as
assessed by the incorporation of [3H]leucine to
trichloroacetic acid-precipitable material. When cycloheximide (2 µg/ml) was added from 13 to 16 h after the start of hypoxia,
levels of cytoplasmic histone-associated DNA fragments (Fig.
5A) and LDH release (Fig. 5B) remained at basal
levels, whereas addition of cycloheximide at 18-20 h had no protective effect. Lower levels of cycloheximide (0.5 µg/ml), which showed a
lesser effect on overall protein synthesis (about 50% suppression), added 14 h into the period of hypoxia, had a diminished protective effect (Fig. 5, C and D).

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Fig. 5.
Effect of cycloheximide on DNA fragmentation
and LDH release by antisense ORP150 HEK cell transfectants. ORP150
antisense-transfected HEK cells (about 2 × 105 cells)
were exposed to hypoxia for 20 h. Cycloheximide (2 µg/ml) was
added at the indicated times (closed bars; A and
B) or cycloheximide at the indicated concentration was added
14 h after the onset of hypoxia (C and D).
Open bars denote the samples prepared from cells incubated
in hypoxia without the addition of cycloheximide (A and
B). In each experiment, cells were harvested 24 h after
the onset of hypoxia. DNA fragmentation (A and C)
and LDH release into the culture medium (B and D)
were measured as in Fig. 4. Mean ± S.D. is shown
(n = 6 for DNA fragmentation and n = 24 for LDH release).
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Several additional lines of evidence supported the conclusion that
ORP150 antisense transfectants were more susceptible to hypoxic stress.
First, DNA prepared from ORP150 antisense transfectants showed
laddering on agarose gels after 15 h of exposure to hypoxia (Fig.
6A, Antisense). In contrast,
wild-type HEK cell controls showed little change in the pattern of DNA
migration up to the longest time point, 30 h (Fig. 6A, wild
type). Second, morphologic study of ORP150 antisense transfectants
revealed features consistent with apoptotic cell death (Fig.
6B). Fluorescence microscopy 18 h following exposure of
cells to hypoxia, using propidium iodide to visualize DNA, displayed
clumped nuclear chromatin and formation of "apoptotic bodies" in
ORP150 antisense transfectants (Fig. 6B, II-III)
compared with uniform faint DNA staining in wild-type controls (Fig.
6B, I). Third, electron microscopy of ORP150
antisense transfectants subjected to hypoxia for 14-18 h showed
fragmented nuclei with condensed chromatin (Fig. 6C,
II-III) and formation of apoptotic bodies (Fig. 6C,
IV). Wild-type control cells harvested even after 20 h of the
onset of hypoxia displayed normal nuclear morphology (Fig. 6C,
I). Furthermore, transfer of phosphatidylserine to the outer
leaflet of the plasma membrane was shown by the increased binding of
annexin V to antisense-transfected HEK cells 12-16 h after the onset
of hypoxia. In contrast, there was no increase in annexin V binding
observed to wild-type HEK cells (Fig. 6D). Enhanced binding
of annexin V to antisense-transfected HEK cells preceded the increase
in propidium iodide-positive cells (Fig. 6E). The percentage
of propidium iodide-positive cells, representing those undergoing the
final stages of the apoptotic process, reached about 40% at maximum.
Taken together, these data are consistent with apoptosis as the
mechanism of cell death which is accelerated in ORP150 antisense
transfectants.

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Fig. 6.
Morphology of ORP150 antisense HEK cell
transfected following exposure to hypoxia. A, either
wild-type or ORP150 antisense transfectants (105 cells)
were maintained in hypoxia for up to 30 h. At the indicated times,
cells were harvested, and DNA was extracted and subjected to agarose
gel (2%) electrophoresis. DNA was visualized by ethidium bromide
staining and ultraviolet light transillumination. B, either
wild-type (B-I) or antisense-transfected HEK cells
(B-II) were exposed to hypoxia for 18 h, fixed, and
stained with propidium iodide (magnification × 100 for I
and II; × 400 for III). C, either
wild-type (C-I) or antisense-transfected HEK cells
(C-II, -III, and -IV) were exposed to hypoxia for
up to 20 h, fixed, and studied by transmission electron
microscopy. Micrographs were obtained from cells exposed to hypoxia for
20 h (I; wild type) or 16 (II), 18 (II), and 20 h (III) for
antisense-transfected HEK cells (bars = 4 µm).
D and E, cells were stained with either
fluorescein isothiocyanate-labeled annexin V (D) or
propidium iodide (E), and positive cells were counted as
described in text. Values are expressed as the percentage of cells
staining positively among the total population of adherent cells (about
100 cells) by the phase contrast microscopic examination. Note that in
D--E, closed bars denote wild-type
HEK cells and open bars denote antisense ORP150-transfected
HEK cells. Mean ± S.D. is shown (n = 6).
ND, not determined.
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Effect of Hypoxia on Energy Metabolism in ORP150
Antisense-transfected HEK Cells--
Depletion of high energy
phosphate compounds, such as ATP, has been suggested as a contributory
factor for hypoxia-induced cell death. ATP content was well maintained
in wild-type and ORP150 antisense transfectants during the first
12 h of hypoxia and slowly decreased, in parallel, over 16-20 h
(Fig. 7). In contrast, once the apoptosis
was triggered in ORP150 antisense transfectants, by 24 h (see Fig.
3A), ATP levels became undetectable in the nonviable cells
(Fig. 7). These data suggest that depletion of high energy metabolites
is not the trigger for accelerated cell death in ORP150 antisense
transfectants.

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Fig. 7.
ATP levels in wild-type and
antisense-transfected HEK cells exposed to hypoxia. Either
antisense transfectants (open bars) or wild-type
(closed bars) HEK cells were exposed to hypoxia (0-36 h),
and ATP content was measured by reversed phase high pressure liquid
chromatography as described in text. Mean ± S.D. is shown
(n = 6). ND denotes the time point at which
ATP content could not be determined due to the cell death.
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Caspase Activity in Hypoxic Wild-type HEK Cells and ORP150
Antisense Transfectants--
To characterize further hypoxia-mediated
cell death in antisense transfectants, apoptosis was induced in both
wild-type HEK cells and antisense ORP150 transfectants by exposure to
staurosporine (1 µM; see Ref. 29). Staurosporine
increased levels of cytoplasmic DNA fragments (Fig.
8A), as well as release of LDH
activity into culture supernatants (Fig. 8B) in both
wild-type and antisense-transfected HEK cells. This was preceded by an
increase in caspase-like-1 and -3 activities in ORP150 antisense
transfectants (Fig. 9, A and
C). In contrast, exposure to hypoxia caused no apparent
increase in these caspase activities (Fig. 9, B and
D). Furthermore, hypoxic HEK antisense transfectants did not
display increased cytosolic cytochrome c antigen (Fig.
9E), although apoptosis was proceeding based on evidence of
DNA fragmentation and morphologic criteria (Fig. 6). However,
staurosporine-treated HEK cells, both wild-type and ORP150 antisense
transfectants, showed an increase in cytoplasmic cytochrome
c (Fig. 9F).

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Fig. 8.
Induction of apoptosis by staurosporine.
Either wild-type (closed bars) and antisense-transfected HEK
cells (open bars) were cultured in the presence of
staurosporine (1 µM). At the indicated times, DNA
fragmentation (A) and release of LDH activity (B)
was measured as described in text. Note that all values of each group
at time "0" in A are equal to 1.0 according to the
formula described in text. Mean ± S.D. is shown
(n = 6 for DNA fragmentation and n = 24 for LDH release).
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Fig. 9.
Caspase-like activity and cytosolic
cytochrome c release during the process of
apoptosis. A-D, wild-type (closed bars) or
antisense-transfected HEK cells (open bars) were incubated
in the presence of either staurosporine (A and C)
or exposed to hypoxia (B and D).
Caspase-1-like activity (A and B) and
caspase-3-like activity (C and D) was assessed
colorimetrically at the indicated times as described in text.
E and F, cell death was induced in either
wild-type (upper panel) or antisense-transfected HEK cells
(lower panel) by exposure to hypoxia (E) or in
the presence of staurosporine (1 µM; F). At
the indicated times, cells were harvested and cytosolic cytochrome
c antigen was assessed by Western blotting with anti-human
cytochrome c antibody. In each experiment, mitochondrial
fraction was also subjected to Western blotting as a positive control
(lane labeled Mt).
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Furthermore, although cleavage of the caspase substrate
poly(ADP-ribose) polymerase, caspase-2, caspase-7, and caspase-9 was clearly observed in staurosporine-induced apoptosis, no such
activation was observed in the hypoxia-mediated cell death in ORP150
antisense-transfected HEK cells (Fig.
10 A-C and E).
Activation of caspase-8 was not observed in antisense-transfected HEK
cells, either cultures exposed to hypoxia or those treated with
staurosporine (Fig. 10D). The activated form of this caspase
was not detected even after the prolonged exposure of the membrane to
the film. These data suggest that hypoxia-mediated cell death in the
antisense ORP150 transfectants involved, at least in part, distinct
pathways from those observed in mitochondria-initiated cell death
following exposure of HEK cells to staurosporine (31).

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Fig. 10.
Activation of caspases in response to
staurosporine or hypoxia in antisense ORP150-transfected HEK
cells. Antisense ORP150-transfected HEK cells (about
106 cells) were either exposed to hypoxia or treated with
staurosporine for the indicated period, and cells were then lysed in
PBS containing Nonidet P-40 (2%), SDS (0.5%), and deoxycholic acid
(0.5%). Protein extracts (10 µg/lane) were applied to
SDS-polyacrylamide gel electrophoresis, followed by the Western
blotting using either goat anti-poly(ADP-ribose) polymerase
(A), goat anti-caspase-2 antibody (B), goat
anti-caspase-7 antibody (C), mouse anti-caspase-8 antibody
(D), or rabbit anti-caspase-9 antibody (E).
Proform and processed forms of these enzymes are indicated by
open and closed arrowheads, respectively. The
migration of simultaneously run molecular weight markers is indicated
on the left side of the gel.
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DISCUSSION |
Expression of ORP150, a stress protein originally purified and
cloned from cultured rat astrocytes subjected to hypoxia, has been
observed in several types of cells subjected to oxygen deprivation (9,
10, 13). Factors responsible for ORP150 expression are also present in
atherosclerotic lesions and in breast cancers. In atherosclerosis,
mononuclear phagocytes infiltrating vascular lesions stained for ORP150
and lipid loading increased ORP150 levels in cultured monocyte-derived
cells (13). In breast cancer, tumor cells at the rapidly growing
periphery of lesions displayed high levels of ORP150, regardless of
estrogen receptor status of the patients, suggesting a role of this
stress protein for cancer cells to invade the less vascular area (17).
Constitutive ORP150 expression is noted in tissues with a well
developed endoplasmic reticulum required to synthesize large amounts of
secretory proteins (e.g. liver and pancreas) (10). This is
similar to what has been reported for in GRP78 and GRP94 (32) and
is consistent with a possible role for ORP150 as a molecular chaperone
and participant in protein folding.
The localization of ORP150 in the endoplasmic reticulum, along with
other polypeptides whose expression is increased in hypoxia, such as
GRP94, and GRP78/Bip, suggests that hypoxia may be a situation characterized by an endoplasmic reticulum stress response (33). Consistent with an important role for GRP78, the most abundant member
of this family expressed constitutively in the endoplasmic reticulum,
homologous recombination experiments in yeast to delete the
GRP78 gene (34) as well as manipulation of GRP78 levels (30)
have shown this factor to have a central role in cellular homeostasis
under physiologic conditions (i.e. in normoxia without other
metabolic stress) and in the cellular response to environmental stress.
The results of our studies with HEK cells stably transfected with an
antisense ORP150 construct, the latter resulting in suppression of
ORP150 expression, provide insights into the contribution of this
polypeptide to cellular properties. Whereas antisense
ORP150-transfected HEK cells proliferate and maintain their viability
under normoxic conditions, following exposure to hypoxia, cell death
increases compared with control cells (wild-type and ORP150
sense-transfected HEK cells). Furthermore, although diminished
expression of ORP150 potentiated hypoxia-induced cytotoxicity,
antisense ORP150-transfected HEK cells did not show increased
vulnerability to other stresses such as sodium arsenate and hydrogen
peroxide. Suppression of ORP150 in antisense HEK transfectants appeared
to be selective, as hypoxia-mediated up-regulation of GRP78 and GRP94
was still observed.
When HEK cells with suppressed ORP150 expression were exposed to
hypoxia, they demonstrated apoptosis based on a number of criteria as
follows: (a) transfer of phosphatidylserine to the outer
leaflet of the plasma membrane; (b) nuclear condensation and
nuclear disorganization (the latter including disruption of nucleoli
and margination of chromatin in discrete masses along the inner side of
the nuclear membrane); (c) DNA fragmentation; and
(d) inhibition of cell death by the addition of
cycloheximide (although the specificity of using cycloheximide to
identify apoptosis is limited, see Ref. 35). However, events leading to
the final common pathway of DNA fragmentation in ORP150
antisense-transfected HEK cells subjected to hypoxia differ from those
in normoxic HEK cells incubated with staurosporine (25). Although
release of mitochondrial cytochrome c antigen into the
cytosol increased caspase-like proteinase activity, and the cleavage of
major caspases are central events in apoptosis associated with
mitochondrial dysfunction (36-38), neither was observed in hypoxic
antisense ORP150 HEK transfectants undergoing apoptosis. These
data suggest that hypoxia-mediated cell death in the ORP150
antisense-transfected HEK cells may involve distinct pathway(s) from
those previously associated with mitochondria-initiated cell death.
Retention of immature/unfolded protein in the endoplasmic reticulum can
cause apoptosis in several cell lines. The accumulation of dengue virus
protein in the endoplasmic reticulum, for example, results in cell
death in a mouse neuronal cell line (39). Furthermore, in HEK cells,
accumulation of immature protein in the endoplasmic reticulum has been
shown to trigger cell death by activation of the transcriptional factor
NF-
B (33). This led us to consider whether the apoptosis-like cell
death observed in the ORP150 antisense HEK transfectants exposed to
hypoxia might be associated with NF-
B activation. However, no
activation of NF-
B was noted in hypoxic HEK cells (either wild-type
or antisense ORP150 transfectants), and addition of the inhibitor
pyrrolidine dithiocarbamate (40) was without effect in our experimental
system. In contrast, pilot data concerning free Ca2+ in HEK
cells indicate impaired buffering of endoplasmic reticulum stores in
the antisense ORP150 transfectants (data not shown), suggesting
potential dysfunction of the endoplasmic reticulum in the ORP150
antisense-transfected HEK cultures.
Low ambient oxygen concentration forces cells to express a set of
stress proteins, thereby facilitating vital functions of cellular
organelles. We have demonstrated that one of those stress proteins, ORP150, is likely to subserve a cytoprotective role at the
level of the endoplasmic reticulum; ORP150 enhances cellular ability to
sustain oxygen deprivation. Analysis of functional properties of ORP150
is likely to add to our understanding of how the cellular response to
hypoxia buttresses intracellular mechanisms enhancing survival due to
environmental stress.