From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Received for publication, May 10, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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Members of the phospholipid scramblase
(PLSCR) family play active roles in altering lipid asymmetry at the
plasma membrane including phosphatidylserine (PtdSer) exposure on the
cell surface. To determine whether PtdSer biosynthesis and
externalization are altered by PLSCR activities during apoptosis,
Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1
and PLSCR2 were established. PLSCR1 was localized on the plasma
membrane, whereas PLSCR2 was predominantly in the nucleus. Cells
overexpressing PLSCR1 showed suppressed growth, altered cell
morphology, and higher basal levels of cell death. Following UV
irradiation, these cells showed earlier and enhanced PtdSer exposure,
increased caspase-3 activation, apoptotic nuclear changes, and PARP
cleavage indicative of apoptosis. UV irradiation in cells
overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis
(accompanied by increased movement of newly made PtdSer into
microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer
formation in UV-irradiated vector control cells increased only by
2-fold. No differences in these responses were observed between
PLSCR2-expressing cells and vector controls. PtdSer synthesis and its
transbilayer movement stimulated by PLSCR1 overexpression were blocked
by a caspase inhibitor along with progression of apoptosis. Thus, our
studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer
externalization and synthesis, implying an up-regulation of PtdSer
formation in response to enhanced outward movement of this phospholipid
to the cell surface during apoptosis. PLSCR1 also appears to influence
progression of UV-induced apoptosis and could be a point of regulation
or intervention during programmed cell death.
Phosphatidylserine (PtdSer)1
externalization is observed during
cell activation, aging, and apoptosis (1-3). This membrane lipid
rearrangement may have various roles depending on the cell type and
conditions for mobilization of PtdSer (4). When exposed on the cell
surface during programmed cell death, PtdSer signals the removal of
apoptotic cells to avoid inflammatory reactions (5, 6). Phospholipid
scrambling also leads to inward movement of sphingomyelin (SM), where
it may be hydrolyzed by neutral sphingomyelinase; subsequent production
of ceramide may play an important role in membrane blebbing during
apoptosis (7). Mechanisms underlying PtdSer externalization are
unclear, but concomitant inactivation of aminophospholipid translocase
activity and activation of phospholipid scramblase (PLSCR) as a result
of rising concentrations of cytoplasmic calcium are considered to play
a major role (8-10). Aminophospholipid translocase helps to maintain
membrane asymmetry by moving PtdSer and PtdEtn on the outer leaflet
back to the inner membrane bilayer (11-13). Phospholipid scramblase,
when activated, catalyzes bidirectional movement of all of the membrane
phospholipids (14-16).
A family of phospholipid scramblases, including HuPLSCR 1-4 and their
murine orthologs, MuPLSCR 1-4, as well as a rat PLSCR homolog to
HuPLSCR1, has been identified (17, 18). PLSCR proteins have a short
extracellular domain or no extracellular domain, whereas their
intracellular domains are highly variable in length and composition
(18). Expression of PLSCR1 is induced at the transcriptional level by
interferon, indicating its potential involvement in interferon-mediated
activities (19, 20). The PLSCRs are type II transmembrane proteins that
are highly conserved in the calcium-binding C-terminal domain (21).
Calcium binding to the proteins is presumed to induce a major
conformational change and activate the lipid scrambling activity (22).
Some members contain PXXP and PPXY motifs,
indicating potential interaction with signaling molecules that contain
SH3 or WW domains (17, 23). PLSCR1 binds to the SH3 domain of c-Ab1
tyrosine kinase and is constitutively tyrosine phosphorylated by this
enzyme (24). PLSCR1 is also found to be enriched in lipid rafts and
phosphorylated upon its interaction with epidermal growth factor
receptor (25). Other post-translational modifications of PLSCR
implicated in regulating its normal functions include palmitoylation at
multiple cysteine residues (26) and phosphorylation at one threonine site through protein kinase C Our previous study using U937 cells showed that PtdSer biosynthesis
increased markedly along with the exposure of PtdSer on the cell
surface during apoptosis induced by various stimuli. Compared with
other newly synthesized phospholipids, PtdSer moved preferentially into
microvesicles budding from apoptotic cells. This stimulation of
synthesis is regulated in a caspase-dependent manner (28).
Normally, PtdSer synthesis occurs at the endoplasmic reticulum and
mitochondria-associated membranes through base exchange of
L-serine with the head group of other existing
phospholipids (29), catalyzed by two isoforms of PtdSer synthase (PSS)
with different substrate specificities. PSS I utilizes
phosphatidylcholine, whereas PSS II converts PtdEtn to PtdSer (30).
Newly synthesized PtdSer is then rapidly transported to other membranes
including the plasma membrane. PtdSer also is delivered to mitochondria where it can be decarboxylated to form PtdEtn. Regulation of PtdSer biosynthesis and its movement in mammalian cells is not well
understood. In CHO-K1 cells, feedback control appears to regulate
serine base exchange reactions to maintain constant levels of PtdSer.
The cells incorporate exogenous PtdSer rapidly, blocking de
novo synthesis of PtdSer by a competitive feedback mechanism
(31-33).
We hypothesized that PtdSer exposure on the cell surface and subsequent
movement into microvesicles may increase the need for more PtdSer as
low intracellular PtdSer levels release feedback inhibition of PtdSer
biosynthesis in response to the PtdSer scrambling activity. To examine
a possible regulatory role of PLSCR, we established CHO cell lines
overexpressing PLSCR1 and PLSCR2 isoforms and monitored biosynthesis of
serine-derived phospholipids and their movement into the vesicles
during UV-induced apoptosis. Our studies suggest that PLSCR activity
influences both synthesis and reorientation of PtdSer in an
isoform-specific and caspase-dependent manner during apoptosis.
Materials--
Anti-c-Myc mAb was purchased from
Clontech. Anti-human poly(ADP-ribose) polymerase
(PARP) pAb was from Santa Cruz Biotechnology. Anti-ACTIVE®-caspase-3
pAb was from Promega. LipofectAMINE 2000 was obtained from Invitrogen.
Annexin-V-FLUOS staining kit was from Roche Molecular Biochemicals.
Trypan blue solution and propidium iodide (PI) were obtained from
Sigma. z-VAD-fmk was purchased from Calbiochem.
L-[3H(G)]Serine,
[methyl-3H]choline chloride, and
[1,2-14C]ethanolamine hydrochloride were obtained from
Mandel Scientific.
Cell Culture--
Strain CHO-K1 was obtained from the American
Type Culture Collection. The cells were maintained in a 5%
CO2 atmosphere in Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 5% fetal bovine serum (CANSERA) and 300 µM proline.
Induction of Apoptosis by UV Irradiation--
Cells grown in
regular growth medium were rinsed with and reseeded in fresh
Dulbecco's modified Eagle's medium with different modifications. The
cells were exposed to a germicidal lamp providing predominantly 254-nm
UV-C light (Philips TUV G30T8 30 W bulb) for 10 min and cultured for
different periods of time.
Cloning of Phospholipid Scramblases into pcDNA3.1/Myc-His(+)
A Expression Vector--
Murine PLSCR1 cDNA (GenBankTM accession
number AF159593) was kindly provided by Dr. Peter Sims (17, 34). Murine
PLSCR2 homolog cDNA was obtained from ATCC expressed sequence tag
clone 1191848, and DNA sequencing indicated that all amino acids were the same as those deposited in GenBank except for a highly conserved Arg-Lys transition at position 178 (GenBankTM accession number AF015790) (21). EcoRI and XhoI sites were
engineered into the 5'- and 3'-ends of the full coding sequences of
PLSCR1 by PCR amplification with primer 1, 5'-GACGAATTCATGGAAAACCACAGCAAGCAAACT-3', and primer
2, 5'-GCACTCGAGCTGCCATGCTCCTGATCTTTGCTC-3'. NotI
and ApaI sites were added to the 5'- and 3'-ends of the full
coding sequences of PLSCR2 by PCR amplification with primer 1, 5'-GACGCGGCCGCATGGAGGCTCCTCGCTCAGGAACA-3', and primer 2, 5'-GCAGGGCCCCTCACAGCCTTCAAAAAACATGTA-3'. PCR-amplified DNA
fragments were cloned into the TA cloning vector pCR2.1-TOPO (Invitrogen) and checked by DNA sequencing. Full coding sequences were
subcloned into the pcDNA3.1/Myc-His(+) A expression vector (Invitrogen) to generate the expression plasmids pcDNA-PLSCR1 and
pcDNA-PLSCR2.
Transient Transfection of CHO-K1 Cells--
CHO-K1 cells grown
to 80-90% confluence were transfected with pcDNA-PLSCR1 or
pcDNA-PLSCR2 constructs (1 µg of plasmid DNA) using LipofectAMINE
2000 reagent according to the manufacturer's instructions. The
transfected cells were grown for 24 h before further analysis.
Establishment of Stable Expression Clones--
To increase the
effectiveness of selection, CHO-K1 cells were co-transfected with empty
pTK-Hyg vector (Clontech) and pcDNA-PLSCR1 or
pcDNA-PLSCR2 constructs according to the manufacturer's
instructions. One day after LipofectAMINE 2000 transfection, selection
medium containing 350 µg of G418/ml and 200 µg of hygromycin B/ml
(Invitrogen) was added to the cells, and the medium was refreshed every
48 h after the initial addition of antibiotics. Cell death was
observed by 3 days after adding antibiotics. Stable clones were
obtained by dilution subcloning and characterized by immunofluorescence and Western blotting. The cells overexpressing PLSCR1, PLSCR2, and
vector control clone were maintained in regular growth medium containing 350 µg of G418/ml and 200 µg of hygromycin/ml.
Western Blotting--
Cells were washed with cold Tris-buffered
saline (TBS). For PARP preparations, the cell extracts were collected
by lysing 1 × 107 cells in 200 µl of sample buffer
(62.5 mM Tris-HCl, pH 6.8, 1.25% SDS, 3.3%
Trypan Blue Exclusion Assay--
The cells were exposed to UV
light for 10 min and cultured for 8 h. The attached cells were
released using 0.25% trypsin and combined with the culture medium
containing floating dead cells. The cells were pelleted by
centrifugation at 1,000 × g for 10 min and were
resusupended in PBS in the presence of an equal volume of trypan blue
solution (0.4%, w/v); the percentage of blue cells/total cells was
counted using a hemocytometer.
Immunofluorescence and Confocal Microscopy--
The cells were
grown on glass coverslips. After incubation, the cells were fixed with
formaldehyde (3%, v/v) and permeabilized with 0.05% (w/v) Triton
X-100 in PBS for 10 min at Labeling Phospholipids with Radioactive Precursors, Lipid
Extraction, and Phosphorus Assay--
After incubation of cells with
20 µCi of [3H]serine, 3 µCi of
[3H]choline, or 2 µCi of
[14C]ethanolamine per dish for various times, the culture
medium was removed and saved. The cells were rinsed twice with 1 ml of cold PBS, and both washes were combined with the original culture medium. The cells were harvested by scraping in 3.5 ml of
methanol:water (5:4, v/v). The lipids were extracted from cell pellets
and medium using a modified Folch procedure (35, 36). Radioactivity in lipid extracts was quantitated using a liquid scintillation counter. Phospholipids were separated using TLC with a solvent system of chloroform:ethanol:triethylamine:water (40:50:40:10, v/v/v/v) and
quantitated with a Bioscan 200 Imaging Analyzer. Total phospholipid mass was determined by measuring phosphorus content of the lipid extracts (37). Total phospholipid biosynthesis was normalized to total
phosphorus in the lipid extracts. The data from three or four
experiments were expressed as the means ± S.E., and the statistical differences were calculated using Student's t test.
Assay for PtdSer Externalization--
Cells grown on glass
coverslips were rinsed with PBS. Annexin-V-fluorescein isothiocyanate
and PI staining was performed according to the manufacturer's
instructions. After rinsing coverslips with binding buffer to remove
unbound annexin-V and PI, the cells were fixed with 4%
paraformaldehyde for 15 min and rinsed twice with PBS. The coverslips
were mounted on glass slides. For the same area of each sample, a
differential interference contrast image (for total cells), a green
fluorescent image (for annexin-V-positive cells), and a red fluorescent
image (for PI-positive cells) were acquired with the Zeiss LSM-510;
superimposed images were obtained with LSM-510 Image software.
Assay for PtdSer Synthase Activity--
After removal of the
culture medium, the cell lysate was prepared by scraping cells in 0.5 ml of ice-cold suspension buffer (250 mM sucrose, 10 mM HEPES buffer, pH 7.5) and sonicating for 30 s on
ice. The samples were centrifuged at Generation and Characterization of CHO-K1 Cell Lines Stably
Overexpressing Murine PLSCR1 or PLSCR2--
Previous studies indicate
that endogenous PLSCR1 cannot be detected in CHO-K1 cells by Western
blotting, and no scramblase activity is detected by in vitro
enzyme assays (27). CHO-K1 cells were transfected with pcDNA-PLSCR1
or pcDNA-PLSCR2 constructs and clones stably overexpressing PLSCR1
and PLSCR2 were then selected. Initially, selection using G418 alone
failed to produce positive clones, raising the possibility that cells
overexpressing PLSCR were outgrown by cells where only the neomycin
resistance gene was incorporated. We then used a strategy to select
G418 and hygromycin B-resistant cells by co-transfecting cells with
vector containing PLSCR and an empty vector containing a hygromycin
resistance gene. With this approach, several G418/hygromycin-resistant
clones were isolated. Introduction of a C-terminal Myc tag ensured that
the overexpressed proteins could be detected by Western blotting and immunofluorescence using an anti-c-Myc mAb. A single protein band was
detected in stable expressing clones transfected with pcDNA-PLSCR1 or PLSCR2 constructs, and both Myc-tagged-PLSCR isoforms migrated with
predicted molecular weights. No endogenous protein from clones transfected with empty pcDNA3.1 vectors was recognized by
anti-c-Myc mAb (Fig. 1A).
Immunofluorescence analyses of these cells showed a predominantly
plasma membrane localization of PLSCR1 cells (Fig. 1B,
middle panel); in some cells, highly expressed PLSCR1 also was seen in the cell nucleus. Membrane vesiculation also was observed in cells overexpressing PLSCR1 (Fig. 1B, middle
panel, arrows). Overexpressed PLSCR2 was found
predominantly in the nucleus but also was apparent in the cytoplasm
(Fig. 1B, right panel). Low background staining
in vector control cells confirmed the high specificity of anti-c-Myc
mAb toward overexpressed PLSCR proteins (Fig. 1B, left
panel). Collectively, our data indicated that CHO-K1 clones
stably overexpressing c-Myc-tagged PLSCR1 and PLSCR2 were generated and
that the levels of expression and subcellular localization of the
introduced proteins could be readily detected with anti-c-Myc mAb.
PLSCR-expressing clones with the best expression rate were used in the
following experiments.
Suppression of Cell Growth and Facilitation of UV-induced Apoptosis
in CHO-K1 Cells by PLSCR1 Overexpression--
When cells were seeded
at same density and monitored for 5 days for their growth rate, vector
control cells doubled every 24 h similar to the wild type CHO-K1
cells, whereas cells overexpressing PLSCR1 grew at a slower rate; by
day 4, cell growth was inhibited by 70% compared with vector control
cells (Fig. 2A).
The shape of PLSCR1-expressing cells was significantly different from
that of wild type CHO-K1 cells, the former being rounder and smaller (Fig. 2B, left panels). PLSCR2-expressing cells
were larger than vector control cells and had a similar growth rate
from day 1-3. After reaching confluence (day 4 or 5), contact
inhibition between PLSCR2-expressing cells resulted in a decreased
growth rate compared with the vector control cells.
When apoptosis was induced in cells overexpressing PLSCR1 or PLSCR2 by
exposing them to UV light for 10 min, cells overexpressing PLSCR1
developed much earlier signs of apoptotic morphology than UV-treated vector cells. Six hours after UV irradiation, when a
majority of control cells with empty vector showed little signs of
apoptosis, the PLSCR1-expressing cells became strongly light reflecting, and cell shrinkage was obvious (Fig. 2B,
right panels). PLSCR2-expressing cells did not show major
differences in developing apoptotic cell morphology compared with
vector cells. When cell death was monitored with a trypan blue
exclusion assay, PLSCR1-expressing cells showed a higher basal level of
cell death, and UV treatment resulted in 30% dead cells, whereas no
significant cell death was observed in UV treatment of vector cells
8 h after UV irradiation (Fig. 2C).
To detect pro-caspase-3 activation as a marker for the progression of
apoptosis, antibody that only recognizes active caspase-3 was used to
detect caspase activation in PLSCR1-expressing and vector cells (Fig.
3A). A higher basal level of
cell death mediated by PLSCR1 overexpression was confirmed because
about 4% of the cells showed positive staining of active caspase-3
(Fig. 3B). Extensive activation of caspase-3 (65%) was
observed in PLSCR1 cells, whereas only a few vector cells (15%) showed
positive signals of activated caspase-3 following 8 h of
incubation after UV irradiation. Nuclear condensation and fragmentation
also were observed in apoptotic cells that showed positive staining for
caspase-3 activation. No differences in caspase-3 activation and
nuclear morphology were observed with PLSCR2-expressing cells compared
with UV-treated vector cells. Thus, PLSCR1 overexpression conferred
growth suppression and promoted UV-induced apoptosis in CHO-K1 cells,
whereas PLSCR2 protein did not have similar effects.
Increased PtdSer Exposure in PLSCR1-expressing Cells Induced with
UV Light--
PLSCR1 is key in mobilizing phospholipids at the plasma
membrane. Overexpression of PLSCR1 in CHO-K1 cells resulted in a
slightly higher basal level of PtdSer externalization, whereas PtdSer
on the cell surface remained low in vector control cells without UV
irradiation (Fig. 4A). When
apoptosis was induced with UV irradiation, PtdSer levels on the surface
of PLSCR1 cells increased as early as 3 h after treatment (data
not shown). By 8 h, the majority of PLSCR1 cells showed positive
annexin-V-fluorescein isothiocyanate stain, indicating major PtdSer
externalization, whereas UV-treated vector cells showed minor changes
(Fig. 4). The integrity of cell membranes was maintained, and few cells
were stained with propidium iodide in their nuclei. PLSCR2-expressing
cells did not show major differences in exposure of PtdSer compared
with vector cells with or without UV treatment (Fig. 4B).
PtdSer externalization also was greatly inhibited by the presence of
z-VAD-fmk both in UV-irradiated PLSCR1-expressing and vector cells
(Fig. 4). Thus, PLSCR1 overexpression facilitated apoptosis and
increased PtdSer externalization in CHO-K1 cells after UV irradiation
in a caspase-dependent manner, whereas PLSCR2
overexpression did not appear to enhance PtdSer externalization or cell
death.
Stimulation of PtdSer Biosynthesis in PLSCR1-expressing Cells
Following UV-induced Apoptosis--
Our previous study with U937 cells
showed that de novo synthesis of PtdSer was stimulated
2-3-fold during programmed cell death induced by various stimulators
of apoptosis (28). Our preliminary experiments indicated that similar
stimulation of PtdSer biosynthesis also was observed in CHO-K1 cells
during UV-induced apoptosis. To test the hypothesis that PtdSer
biosynthesis and externalization to the cell surface were related
events and that increased outward movement of PtdSer may further
increase PtdSer biosynthesis, we measured the biosynthesis of PtdSer
and other serine-derived phospholipids using cells overexpressing
PLSCR1 or PLSCR2. After UV irradiation of cells followed by a 12-h
incubation with [3H]serine, there was a 2-fold increase
in PtdSer synthesis in vector cells compared with those without UV
treatment (Fig. 5, top panel). PLSCR2-expressing cells had basal and UV-stimulated rates of PtdSer biosynthesis similar to control cells. Without any UV irradiation, the
basal rate of PtdSer synthesis was slightly higher (1.7-fold) in cells
overexpressing PLSCR1. With UV stimulation there was a significant
increase in PtdSer biosynthesis in cells overexpressing PLSCR1; PtdSer
biosynthesis was 4-fold higher compared with untreated PLSCR1-expressing cells. Although PtdSer decarboxylation to PtdEtn slightly decreased following UV irradiation in vector and
PLSCR2-expressing cells, decarboxylation was increased slightly in
PLSCR1-expressing clones, indicating increased transport of newly
synthesized PtdSer into the mitochondria (Fig. 5, middle
panel). SM biosynthesis was stimulated 1.5-fold in UV-irradiated
PLSCR1 or PLSCR2-expressing cells and 2.5-fold in vector cells
following UV irradiation (Fig. 5, bottom panel). A higher
amount of newly synthesized PtdSer also was recovered from medium
(representing mainly vesicles released during apoptosis) with
UV-treated PLSCR1-expressing cells compared with vector controls (Fig.
6).
In vitro serine base exchange assays were performed to test
the possible involvement of altered PtdSer synthase levels in regulating PtdSer biosynthesis in cells overexpressing PLSCR1 (Table
I). PLSCR1-expressing cells showed 30%
higher serine base exchange activities compared with vector cells. UV
treatment did not change the specific activities of PtdSer synthase
enzymes in these cells. A 20% decrease was detected in UV-treated
vector cells compared with untreated vector cells.
De novo biosynthesis of phosphatidylcholine monitored with
radiolabeled choline was inhibited in PLSCR1-expressing cells by 25%
following UV treatment, whereas no change was observed in UV-treated
control cells. PtdEtn biosynthesis from radiolabeled ethanolamine was
inhibited slightly in control cells and unchanged in PLSCR1 cells with
UV treatment (Table II). Thus,
overexpression of PLSCR1 cells resulted in a significant increase in
PtdSer biosynthesis following UV-induced apoptosis, and newly
synthesized PtdSer was released at a higher rate into medium as
microvesicles.
Caspase Dependence of PtdSer Biosynthesis in PLSCR1-expressing
Cells--
Stimulation of PtdSer biosynthesis in wild type CHO-K1
cells during UV-induced apoptosis was independent of caspase activation and was not blocked by z-VAD-fmk, a general caspase inhibitor (data not
shown). Cells overexpressing PLSCR1 showed significant basal levels of
PARP cleavage, a marker for caspase-3 activation. When 100 µM z-VAD-fmk was added to cells after UV irradiation, apoptosis was blocked in both vector and PLSCR1-expressing cells, based
on a lack of PARP cleavage (Fig.
7A). Stimulation of PtdSer biosynthesis was unchanged in the presence of z-VAD-fmk in UV-treated vector cells (similar to wild type CHO-K1 cells). PtdSer biosynthesis stimulated in PLSCR1-expressing cells was inhibited by z-VAD-fmk to a
level similar to that of the UV-treated vector cells, indicating that
further stimulation of PtdSer biosynthesis as a result of PLSCR1
overexpression was blocked by z-VAD-fmk (Fig. 7B). Movement of newly synthesized PtdSer into microvesicles was sensitive to z-VAD-fmk in both vector and PLSCR1-expressing cells (Fig.
7C). z-VAD-fmk showed minimal effects on PtdSer
decarboxylation in vector and PLSCR1-expressing cells treated with or
without UV irradiation. UV-stimulated SM synthesis in vector cells was
not changed by z-VAD-fmk, but the caspase inhibitor further increased SM stimulation in UV-treated PLSCR1-expressing cells to the level similar to that of the UV-treated vector cells (data not shown). Thus,
stimulation of PtdSer biosynthesis in PLSCR1 cells was partially dependent on caspase activation, but caspase-independent mechanisms were involved in up-regulating PtdSer biosynthesis in vector cells after induction of apoptosis by UV exposure.
Externalization of PtdSer on the cell surface is a key signal for
removal of apoptotic cells. Our previous studies showed that
transbilayer migration of PtdSer was correlated with PtdSer synthesis
by a caspase-dependent process (28). To test the postulated relationship between PtdSer externalization and its biosynthesis, we
studied the involvement of PLSCR isoforms responsible for active translocation of PtdSer to the cell surface in regulating PtdSer biosynthetic pathways. Stable overexpression clones of two PLSCR isoforms were established in CHO-K1 cells. Because endogenous PLSCR1
cannot be detected in CHO-K1 cells by Western blotting and because no
scramblase activity is detected by in vitro assays (27), we
anticipated that addition of PLSCR to the cells by transfection might
enhance PtdSer synthesis and/or its externalization in response to
induction of apoptosis.
When overexpressed in CHO-K1 cells, PLSCR1 was located predominantly in
the plasma membrane of the cell and, in some cases, in the nuclei.
Palmitoylation at conserved cysteines in PLSCR1 is required for
anchoring this protein to the plasma membrane (26); overexpressed
PLSCR1 proteins not modified by palmitoylation may be moved into the
nuclei. Overexpression of PLSCR1 resulted in changes in cell
morphology, increased basal cell death, and slower rate of cell growth.
Noticeably, PLSCR1-expressing cells also had higher basal levels of
PtdSer expression on the cell surface. Membrane blebbing also was
observed in these cells. When PLSCR1 is stably expressed in ovarian
carcinoma cell line HEY1B (20), no changes in growth rate and
morphology are found in cells grown in serum culture, but significant
suppression of tumor development is observed when PLSCR1-expressing
cells are implanted into athymic nude mice (20). Our data indicate that
PLSCR1 can suppress cell growth in serum culture under different cell
contexts. The implications or mechanisms of morphological changes in
PLSCR1 overexpressing cells are not clear, but increased membrane lipid movement and blebbing may contribute to the observed changes. PtdSer is
found to be preferentially exposed in membrane domains, particularly in
membrane blebs (39), indicating that higher PLSCR1 activity may direct
PtdSer to these surface regions.
When apoptosis was induced by UV irradiation, PLSCR1-expressing cells
developed morphological and biochemical changes much earlier than the
UV-treated vector cells. First, PLSCR1 cells rapidly exposed PtdSer to
the surface; by 8 h following UV stimulation, majority of the
cells overexpressing PLSCR1 showed positive annexin-V binding, whereas
very few control cells had exposed PtdSer. Second, caspase-3 activation
occurred much earlier in PLSCR1 cells compared with controls. Third,
nuclear fragmentation in PLSCR1 cells preceded that in control cells.
Collectively, these dramatic differences indicated that PLSCR1 plays an
important role in promoting PtdSer externalization following induction
of apoptosis. Thus, PLSCR1 can be considered to be anti-proliferative
and pro-apoptotic as overexpression leads to suppression of growth and
extensive cell death in untreated cells and facilitates apoptosis in
UV-induced cells.
To determine relationships between increased PtdSer
movement to the outer surface and synthesis of new PtdSer, lipid
biosynthesis was examined using labeled serine as a precursor. Based on
incorporation and metabolism of labeled serine by intact cells, clones
overexpressing PLSCR1 showed higher basal levels of PtdSer biosynthesis
and externalization compared with vector control cells. In
vitro assays of serine base exchange activity (recognizing that
maximal activities measured under in vitro conditions may
not fully reflect the capacity for synthesis in intact cells within a
localized milieu of in vivo activators or inhibitors)
suggest that an increase in PSS enzyme activity in PLSCR1-expressing
cells may contribute to the higher basal levels of PtdSer biosynthesis
measured in intact cells.
Following UV irradiation of intact cells, the stimulation of PtdSer
biosynthesis measured by labeled serine incorporation was significantly
higher in PLSCR1-expressing cells compared with control cells
containing empty vector, and more newly synthesized PtdSer moved into
microvesicles; apoptotic cells overexpressing PLSCR1 had a 2.5-fold
increase in PtdSer synthesis compared with UV-treated vector cells.
Potentially, an increase in PtdSer level could be explained by a
blockage of PtdSer decarboxylation, but this apparently was not the
case, because synthesis of serine-derived PtdEtn was even slightly
enhanced in PLSCR1-expressing cells following UV irradiation,
indicating increased transport of PtdSer into mitochondria. Also, no
major changes in serine uptake were observed in PLSCR1-expressing cells
and vector cells following UV irradiation (data not shown). Although
not a predicted correlation, specific activities of serine base
exchange enzymes measured in vitro remained unchanged in
apoptotic PLSCR1-expressing cells upon UV treatment, indicating that an
increase in the cellular content of base exchange enzymes may not be
the primary reason underlying the stimulation of PtdSer biosynthesis
observed in both cell lines. This implies that changes in cellular
PtdSer biosynthesis during triggering of apoptosis involves more than
an increase in the amount of available enzyme. As one explanation of
this unanticipated observation, we propose that activation of PtdSer
externalization to the cell surface of intact cells, facilitated
through overexpression of PLSCR1, may relieve PtdSer feedback to
enhance base exchange activity and new PtdSer biosynthesis in both
untreated and UV-treated PLSCR1-expressing cells without a change in
actual amount of PSS enzyme. Movement of PtdSer to the outer leaflet of
the plasma membrane and its migration into vesicles may deplete PtdSer
on the inner surface of the membrane bilayer. This may produce a signal
for enhanced PtdSer biosynthesis as PtdSer-mediated inhibition of the
production of new PtdSer is released (Fig.
8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(27). PLSCR proteins not only serve to activate membrane lipid scrambling but also play versatile roles in signaling pathways that regulate cellular events such as
proliferation and oncogenic transformation (20, 24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 12.5% glycerol, 0.05% bromphenol blue). For
scramblase preparations, total protein extracts were prepared by lysing
cells in 0.5 ml of lysis buffer (1% Triton X-100, 40 µl/ml 5×
protease inhibitor mixture (Roche Molecular Biochemicals) in TBS)
followed by incubation on ice for 10 min. The cell lysate was
centrifuged at 15,000 × g for 10 min at 4 °C.
Protein in a 200-µl aliquot was precipitated with 1 ml of cold
acetone. The protein concentration of each sample was determined using
a micro BCA protein assay kit (Pierce). The samples were resolved by
SDS-PAGE (8% for PARP and 10% for Myc-tagged PLSCR1 and PLSCR2) and
transferred to polyvinylidene difluoride membrane (Millipore) according
to the manufacturer's instructions. For PARP antibody blotting, the
membrane was incubated for 1 h with hybridization solution
containing anti-human PARP (1:4,000) in 5% skim milk with TTBS (0.04%
Tween 20 in TBS). The blot was rinsed with TBS and incubated for 45 min
with goat anti-rabbit horseradish peroxidase-coupled secondary antibody
(1:10,000) in 5% skim milk with TTBS. For detection of Myc-tagged
proteins, the membrane was incubated with hybridization solution
containing anti-c-Myc mAb (1:2,000) according to the manufacturer's
instructions. The blot was then incubated with goat anti-mouse
horseradish peroxidase-coupled secondary antibody (1:10,000). Enhanced
chemiluminescence (Amersham Biosciences) was used to detect relevant
proteins according to the manufacturer's instructions.
20 °C. The cells were incubated with
anti-c-Myc mAb (1:500, v/v) in PBS containing 1% BSA (PBS-BSA)
overnight at 4 °C and rinsed with PBS-BSA. Alexa flour
488-conjugated goat anti mouse secondary antibody (Molecular Probes)
was added (2 µg/ml) and incubated for 45 min at 22 °C. The cells
were rinsed twice with PBS-BSA. The coverslips were mounted in 2.5%
1,4-diazabicyclo (2,2,2)octane and 90% glycerol in 50 mM
Tris-HCl (pH 9.0) on glass slides. Detection of activation of caspase 3 was performed with overnight incubation with anti-ACTIVE®-caspase-3 pAb (1:500, v/v) at 4 °C followed by staining with Alexa flour 488 goat anti-rabbit IgG at 22 °C. Propidium iodide was added 30 min
after incubation with the secondary antibody and incubated for another
15 min. Alexa flour 488 staining was visualized with excitation at 488 nm, and PI staining was visualized with excitation at 543 nm using a
Zeiss inverted laser-scanning confocal microscope, LSM-510.
Superimposed images were obtained with LSM-510 Image software.
4 °C at 600 × g for 2 min. The supernatant from cell extracts (100 µl)
was added to an equal volume of prewarmed assay mixture (5 mM CaCl2, 50 mM HEPES buffer (pH
7.5), and 1 µCi of [3H]serine) and incubated at
37 °C for 20 min (38). The reactions were terminated by adding 1.5 ml of methanol:H2O (5:4, v/v) and 2 ml of chloroform. The
samples were mixed thoroughly and centrifuged at 2,000 rpm for 10 min.
Lipid was extracted using a modified Folch procedure, and the total
radioactivity was determined using a liquid scintillation counter.
Protein concentration was measured using a Micro BCA kit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stable expression of c-Myc-tagged PLSCR1 or
PLSCR2 in CHO-K1 cells. A, cells stably overexpressing PLSCR
isoforms and controls transfected with empty pcDNA3.1 vectors were
harvested, and the proteins were extracted and separated (20 µg)
using SDS-PAGE and transferred to polyvinylidene difluoride membrane.
c-Myc-tagged proteins were detected by Western blotting with anti-c-Myc
mAb as described under "Experimental Procedures." B,
immunofluorescence was performed on CHO-K1 clones stably overexpressing
PLSCR1 or PLSCR2 and on vector controls. The cells were fixed,
permeabilized, and incubated with anti-c-Myc mAb. Alexa fluor
488-conjugated goat anti-mouse IgG was used to detect Myc-tagged
proteins. The cells expressing PLSCR1 cells were analyzed by confocal
microscopy at a position highlighting the cell membrane. Membrane
vesiculation was observed at the plasma membrane (arrows).
The image of PLSCR2-expressing cells was taken at the midpoint of the
nucleus. Bar, 10 µm. Vec, vector.
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Fig. 2.
Growth suppression and early occurrence of
UV-induced apoptosis in CHO-K1 cells overexpressing PLSCR1. In
A, CHO-K1 cells expressing PLSCR1, PLSCR2, and control
clones were seeded at 1 × 105 cells/60-mm dish on day
0 and maintained in normal growth medium as described under
"Experimental Procedures." At the time indicated, the cell numbers
were determined in triplicate samples and expressed as the means ± S.E. B and C show cells that were treated
with or without UV light followed by 6 h (B) or 8 h (C) incubations. For B, phase contrast photographs were taken at 300×
magnification. For C, cell death in PLSCR1-expressing cells
and vector controls was assessed using a trypan blue exclusion assay as
described under "Experimental Procedures." The data are the
means ± S.E. from triplicate samples of one representative
experiment. Vec, vector.
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Fig. 3.
Early caspase-3 activation following UV
irradiation in CHO-K1 cells overexpressing PLSCR1. CHO-K1 cells
overexpressing PLSCR1 or PLSCR2 and vector controls were treated with
or without UV light and were cultured for 8 h. The cells were
fixed, permeabilized, and incubated with anti-ACTIVE®-caspase-3 pAb.
Fluorescein-conjugated goat anti-rabbit IgG was used to detect the
activated form of caspase-3 (green). Propidium iodide was
used to stain the nucleus (red). For A, the
images were taken with a confocal microsope as described under
"Experimental Procedures." Bar, 20 µm. For
B, ~1000 cells were counted in each sample to determine
the cells positive for active caspase-3. Vec, vector.
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Fig. 4.
Increase in PtdSer externalization following
UV irradiation in PLSCR1-expressing cells and inhibition of UV-induced
PtdSer externalization by caspase inhibitor. CHO-K1 cells
overexpressing PLSCR1, PLSCR2, and vector controls were treated with or
without UV light and cultured for 8 h. In some cases, z-VAD-fmk
(100 µM) was added to cells as indicated. The cell
surface exposure of PtdSer and membrane integrity were assayed with
annexin-V-fluorescein isothiocyanate (green) and propidium
iodide (red) staining, respectively. A, images
were taken with a confocal microscope as described under
"Experimental Procedures." Bar, 20 µm. B,
~1000 cells were counted for positive staining with annexin-V in each
sample. The values are the means ± S.E. of triplicate samples
from one representative experiment. NT, not tested;
Vec, vector; DMSO, dimethyl sulfoxide.
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Fig. 5.
Biosynthesis of serine-derived phospholipids
in CHO-K1 cells overexpressing PLSCR1 and PLSCR2. CHO-K1 cells
overexpressing PLSCR1 or PLSCR2 and vector controls were seeded in
serine-free Dulbecco's modified Eagle's medium. Following treatment
without (white bars) or with (black bars) UV
light for 10 min, [3H]serine (20 µCi) was added to the
cells. The cells were incubated for 12 h and harvested; lipids
were extracted and separated by TLC. Radioactivity in PtdSer, PtdEtn,
and SM was determined. The data are the means ± S.E. of six
samples. Vec, vector.
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Fig. 6.
Recovery of PtdSer in culture medium
following UV irradiation. Cell treatment and incubation were as
described for Fig. 5. After incubation, the medium was collected, and
the lipids were extracted and separated by TLC. Radioactivity in PtdSer
from medium was determined. The data are the means ± S.E. of six
samples. Vec, vector.
Serine base exchange activities
PtdCho and PtdEtn biosynthesis in cells overexpressing PLSCR1 through
CDP-choline and CDP-ethanolamine pathways
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Fig. 7.
Effects of caspase inhibitor on
PtdSer synthesis in CHO-K1 cells overexpressing PLSCR1 following UV
irradiation. PLSCR1-expressing cells and vector controls were
seeded in serine-free Dulbecco's modified Eagle's medium. Following
treatment of cells without or with UV light, z-VAD-fmk (100 µM) or Me2SO (control) was added to cells as
indicated. For A, the cells were harvested 12 h after
UV irradiation, and the proteins were extracted and separated (20 µg)
using SDS-PAGE. Immunoblotting was performed with anti-human PARP pAb
as described under "Experimental Procedures." For B and
C, the cells were incubated for 12 h in the presence of
20 µCi of [3H]serine. The cells were harvested, and the
lipids were extracted and separated by TLC. Radioactivity in PtdSer was
determined. The values are the means ± S.E. of six samples.
B, total PtdSer from cells and the medium; panel
C, PtdSer from the medium. Vec, vector.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 8.
Proposed effects of PLSCR1 overexpression on
PtdSer biosynthesis and apoptosis. In CHO-K1 cells, overexpression
of PLSCR1 leads to enhanced PtdSer externalization to the cell surface.
This provides signals to release the feedback control of PtdSer
biosynthesis by the product itself. Consequently, de novo
biosynthesis is stimulated. Because PLSCR1-mediated PtdSer exposure
requires the activation of caspases, stimulation of PtdSer biosynthesis
during UV-induced apoptosis also is caspase-dependent.
Overexpression of PLSCR1 in CHO-K1 cells positively regulates the
progression of UV-induced apoptosis through unknown mechanisms.
In contrast to the greater effect on PtdSer synthesis, PLSCR1-expressing cells had only a 1.5-fold increase in SM formation compared with treated control cells and a 2.5-fold stimulation following UV irradiation. It seems that pathways of PtdSer biosynthesis and trafficking are up-regulated to a greater extent in cells overexpressing PLSCR1 even though the SM biosynthesis pathway shares serine as a common substrate.
In both the parent CHO-K1 cells and vector-transfected controls, the 2-fold stimulation of PtdSer synthesis following UV irradiation occurred independently of caspase activities. Other studies in our laboratory indicate that UV-mediated stimulation of PtdSer synthesis in CHO-K1 cells, catalyzed by overexpressed PSS I or PSS II, does not require caspase activation.2 On the other hand, the 4-fold stimulation of PtdSer formation in PLSCR1-expressing cells seems to have both caspase-dependent and caspase-independent components as co-incubation of z-VAD-fmk reduced PtdSer levels in UV-treated PLSCR1-expressing cells back to that of the UV-treated control cells but did not reduce it to the level of untreated cells. The caspase-regulated step appears to be PtdSer externalization mediated by PLSCR1. The serine base exchange reaction per se does not require activation of caspases; it only becomes sensitive to caspase inhibition when its activity is triggered by PtdSer externalization, possibly through indirect release of feedback inhibition (Fig. 8).
When PLSCR2, a shorter isoform of scramblase without an extracellular tail was expressed, the protein was found predominantly inside the nucleus. PLSCR2 overexpression does not facilitate PtdSer externalization or alter PtdSer biosynthesis in CHO-K1 cells during UV-induced apoptosis. This further confirms that PLSCR1-mediated PtdSer externalization leads to the stimulation of PtdSer formation. Mouse PLSCR2 when reconstituted into proteoliposomes in vitro has been reported to catalyze NBD-PC scrambling similar to human PLSCR (21). Possibly, PLSCR2 was not targeted to the right region of our cells, and its presence in the nucleus is inadequate to influence PtdSer externalization and apoptosis.
In summary, our data indicate that PLSCR1, when overexpressed in CHO-K1
cells, promotes externalization of PtdSer at the plasma membrane and
facilitates UV-induced apoptosis. The PLSCR2 isoform is targeted
primarily to the nucleus where its role is not clear. PtdSer
biosynthesis is greatly stimulated in PLSCR1-overexpressing cells
induced to undergo apoptosis. This stimulation is dependent on caspase activation, possibly mediated by feedback by altered levels
of PtdSer on the inner surface of the plasma membrane to increase base
exchange activity without changing levels of the PSS enzyme. Thus,
PLSCR1 at the plasma membrane appears to be a point of control in
regulating the apoptotic process and may mediate a feedback signal that
results in enhanced biosynthesis to replace mobilized PtdSer. Continued
PtdSer synthesis may be required for sustained apoptosis. It is
apparent that the externalization of PtdSer mediated by altered
scramblase activities is a potential point of regulation and hence a
possible target for therapeutic intervention in the complex process of
programmed cell death and removal of dead cells and debris from the
body by immune response systems.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Peter Sims from the Scripps Research Institute for providing the full cDNA sequences of MuPLSCR1 and MuPLSCR2. The skilled technical assistance of Robert Zwicker and Gladys Keddy with cell culture is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by an IWK Health Centre Graduate Student Scholarship (to A. Y.) and Grants MGC-11476 and MT-15283 from the Canadian Institutes for Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Atlantic
Research Centre, 5849 University Ave., Dalhousie University, Halifax, NS B3H 4H7, Canada. Tel.: 902-494-7066; Fax: 902-494-1394;
E-mail: h.cook@dal.ca.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M204614200
2 A. Yu, C. R. McMaster, D. M. Byers, N. D. Ridgway, and H. W. Cook, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PtdSer, phosphatidylserine; CHO, Chinese hamster ovary; mAb, monoclonal antibody; pAb, polyclonal antibody; PARP, poly(ADP-ribose) polymerase; PLSCR, phospholipid scramblase; PtdEtn, phosphatidylethanolamine; SM, sphingomyelin; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone; PSS, PtdSer synthase; PI, propidium iodide; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
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