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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2593-2599
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Transcriptional control of the human plasma membrane
phospholipid scramblase 1 gene is mediated by
interferon-
Quansheng Zhou,
Ji Zhao,
Fahad Al-Zoghaibi,
Aimin Zhou,
Therese Wiedmer,
Robert H. Silverman, and
Peter J. Sims
From the Department of Molecular and Experimental Medicine and
Department of Vascular Biology, The Scripps Research Institute, La
Jolla, California; and the Department of Cancer Biology, Lerner
Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio.
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Abstract |
Interferons (IFNs) mediate their diverse biologic activities through
induction of the expression of multiple genes. Whereas the mode of
action of certain of these IFN-regulated genes has been well
characterized, most of the molecular and cellular events underlying the
constellation of biologic responses to the IFNs remain unresolved. This
study showed that the newly identified PLSCR1 gene for
phospholipid scramblase, previously implicated in remodeling of plasma
membrane phospholipids, is regulated at the transcriptional level by
IFN- . Analysis of 5' flanking genomic sequence in reporter
constructs showed that transcriptional control of PLSCR1 was
entirely regulated by a single IFN-stimulated response element located
in the first exon. A similar induction of PLSCR1 by IFN-2a
was also observed in a variety of other human tumor cell lines as well
as in human umbilical vein endothelial cells. In these cell lines, the
marked IFN-2a-induced increase in PLSCR1 protein expression,
ranging as high as 10-fold above basal levels, was not accompanied by
increased cell surface exposure of phosphatidylserine, suggesting that
remodeling of the cell surface requires both exposure to IFN and a
second yet-to-be identified event to stimulate plasma membrane
phospholipid scramblase activity and to mobilize phosphatidylserine to
the cell surface.
(Blood. 2000;95:2593-2599)
© 2000 by The American Society of Hematology.
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Introduction |
Interferons (IFNs)1 are a family of
cytokines that exert antiviral, antiproliferative, antitumor, and
immune modulatory functions through transcriptional induction of
specific IFN-stimulated genes (ISGs).1,2 On binding to
their cell surface receptors, IFNs initiate a signaling cascade
involving the JAK family of tyrosine kinases and signal transducers and
activators of transcription (STATs) that ultimately results in ISG
transcription. The cellular proteins encoded by ISGs largely mediate
the actions of IFNs. The best characterized ISGs include certain
proteins that are thought to contribute to the antiviral properties of
IFNs, most notably the double-stranded RNA-activated protein kinase
(PKR), RNase L, the 2'-5' oligoadenylate (2-5A)
synthetases, and the Mx proteins, as well as those ISGs that directly
participate in transcriptional regulation of ISG and IFN-gene
expression, including the STATs and IFN regulatory factor (IRF)
families of transcription factors.1,3,4 The biologic
functions of many other ISGs remain unclear and the mechanisms by which
the IFNs exert their diverse range of biologic activities remains
largely unresolved.1,2,5
Phospholipid scramblase 1 (PLSCR1; GenBank Accession Numbers
AF098 642, AF153 715) encodes an endofacial plasma membrane protein that is implicated to mediate an accelerated transbilayer movement of
membrane phospholipids under conditions of elevated calcium or
acidification of the cytosol.6-8 The properties of this
protein suggest that it may contribute to the rapid transbilayer
movement of plasma membrane phospholipids that is observed in activated platelets and injured or apoptotic cells that are exposed to elevated intracellular [Ca++].9-12 Whereas PLSCR1
is detected in a wide range of cells and tissues, it has been noted
that there can be marked cell-to-cell variability in the level of
expression of messenger RNA (mRNA) or protein.7 When
membranes differing in PLSCR1 content were compared, the level of
PLSCR1 protein generally correlated with the observed sensitivity of
membrane phospholipids to the effects of elevated Ca++
at the endofacial surface.8
Differential oligonucleotide array screening of ISG expression in the
IFN-responsive fibrosarcoma cell line HT1080 suggested a nearly 10-fold
increase of PLSCR1 mRNA in cells treated with either IFN- or
IFN- , and to lesser degree in response to IFN- .5 This
study demonstrates that the amount of PLSCR1 expressed in the plasma
membrane of a variety of cells is under transcriptional control by an
IFN-stimulated response element located in the untranslated first exon,
and we consider how this up-regulation of PLSCR1 expression may relate
to the biologic activities of IFN.
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Materials and methods |
Materials
Recombinant human interferon-2a (IFN-2a)
3 × 106 IU/mL was from Roche Laboratories (Nutley, NJ).
Bovine prothrombin, factor Va, and factor Xa were obtained from
Haematologic Technologies (Essex Junction, VT). Chromogenic thrombin
substrate CBS 34.47 was from Diagnostica Stago, Asnières, France.
RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), modified
Eagle's medium essential (MEME), and OPTI-MEM were from GIBCO-BRL
(Grand Island, NY). Murine monoclonal antibody V237 specific for the
light chain of factor Va was a gift from Dr Charles T. Esmon (The
Oklahoma Medical Research Foundation, Oklahoma City, OK).13
Murine monoclonal antibody 4D2 was raised against purified recombinant
human PLSCR1. Cell lines: Daudi, Raji, HeLa, and Jurkat cells were from
American Type Culture Collection (Rockville, MD); human umbilical vein endothelial cells and CS-C medium were from Cell Systems Co. (Kirkland, WA); fibrosarcoma cell lines HT1080 and STAT1-null U3A cells were a
gift from Dr George R. Stark (Cleveland Clinic Foundation, Cleveland, OH).14
Cell culture
The Burkitt's B-cell lymphoma cell lines, Daudi and Raji, and
Jurkat T-cell line were cultured in RPMI-1640 complete medium. Human
fibrosarcoma HT1080 cells and U3A cells were cultured in DMEM, HeLa
cells in MEME, and human umbilical vein endothelial cells in CS-C
medium. All culture media were supplemented with 10% fetal bovine
serum (FBS; 20% in case of Daudi cells) and 100 U/mL of penicillin and
100 µg/mL of streptomycin, and all cells were maintained at 37°C
in 5% CO2.
Northern blotting
Cells were washed twice in phosphate-buffer saline (PBS) and the
total RNA was extracted with Trizol reagent (GIBCO-BRL). RNAs, 20 µg/lane, were separated in 1.2% agarose, 2.2 mol/L formaldehyde gels
and transferred to Nylon membranes (Amersham, Piscataway, NJ) for 18 to 20 hours. RNA was cross-linked to the
membrane, incubated in prehybridization solution at 42°C for 16 hours, and probed with an EcoRI fragment of PLSCR1 complementary
(cDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, or
-actin cDNA labeled with 32P-dCTP by random priming with
the Prime-a-gene labeling system (Promega, Madison, WI). Membranes were
washed and used to expose x-ray film.
Western blotting
For Western blotting, 106 cells were harvested and lysed
in 30 µL of cell lysis buffer (2% NP-40 in PBS containing 5 mM EDTA, 50 mM benzamidine, 50 mM N-ethyl maleimide, 1 mM phenylmethylsulfonyl fluoride, and 1 mM leupeptin) at 4°C for 1 hour. Cell lysate was centrifuged at 250,000g for 30 minutes at 4°C, and the
supernatants denatured (100°C, 5 minutes) in 10% (w/v) sodium
dodecyl sulfate (SDS) sample buffer containing 2% -mercaptoethanol.
Following SDS-polyacrylamide gel electrophoresis (0.23 µg total
protein per lane) and transfer to nitrocellulose, the membrane was
blocked with 4% low-fat milk and incubated for 1 hour at room
temperature in the presence of 2 µg/mL of 4D2, a monoclonal antibody
raised against PLSCR1. The blots were incubated with horseradish
peroxidase-conjugated goat antimouse IgG (Sigma, St Louis,
MO) for 1 hour at room temperature, developed by
SuperSignal ULTRA Chemiluminescence (Pierce, Rockford, IL), and
analyzed on a Kodak Image Station 440CF (Eastman Kodak, Rochester, NY).
PLSCR1 antigen detected by Western blotting was quantified using
Kodak's 1D Image Analysis Software version 3.0.
Protein concentrations
The protein concentration of cell lysates was determined by
bicinchoninic acid (BCA) assay. In brief, 150 µL of 1:90
diluted cell lysate was mixed with 150 µL of BCA reagent (Pierce,
Rockford, IL) and incubated at 37°C for 30 minutes. Absorbance at
562 nm was measured, and protein concentration calculated using bovine serum albumin (BSA) as standard.
Confocal fluorescence microscopy
Cells were subcultured on glass cover slips and treated with 1000 IU/mL of IFN-2a for 0 to 18 hours at 37°C. All following procedures were performed at room temperature. Cells were washed in PBS
and fixed with 2% paraformaldehyde in PBS for 30 minutes. After
permeabilization by 0.005% saponin in PBS for 5 minutes, cells were
incubated in 2% whole goat serum in PBS for 30 minutes, followed by
incubation with mab 4D2 (20 µg/mL in 2% goat serum in PBS) for 1 hour. Cells were stained with fluorescein isothiocyanate (FITC)-goat
antimouse IgG (2µg/mL in PBS) for 1 hour, followed by nuclear
counterstain with propidium iodide (0.1 µg/mL in PBS) for 10 minutes.
Cover slips were mounted on glass slides and samples analyzed on a
Bio-Rad MRC1024 laser scanning confocal microscope attached to a Zeiss
Axiovert S100TV microscope with Infinity Corrected Optics (40 × oil
immersion objective). Images were collected using Bio-Rad's LaserSharp
(v3.2) software. Specificity of staining observed for mab 4D2 was
evaluated by cell staining with the identical concentration of an
isotype-matched antibody raised against complement C9, substituting for
mab 4D2.
Molecular cloning of 5' flanking region of PLSCR1
gene and construction of deletions
Human PLSCR1 gene was cloned from a BAC-human genomic
library (Genome System, St. Louis, MO) using full-length PLSCR1 cDNA for hybridization, and 4.12 kb of 5' flanking region was
sequenced (GenBank AF153 715). The 4.18-kb DNA consisting of the
5' flanking region ( 1 to 4120) and the first 60 bp of the
first exon of the gene (+1 to +60) was amplified by polymerase
chain reaction (PCR) using Advantage DNA polymerase mix (CLONTECH
Laboratories, Palo Alto, CA), and PCR products were cloned into
pGL3-basic-luciferase reporter vector (Promega, Madison, WI). Analysis
of the 5' flanking region for the presence of putative binding
sites for transcription factors was performed using MatInspector V2.2.
The 4 putative binding sites for ISGF3 or IRFs (Figure 5) were deleted
by PCR-mediated deletion. All DNA sequencing was performed on an ABI
DNA Sequencer Model 373 Stretch (Applied Biosystems, Foster City,
CA) using PRISM Ready Reaction DyeDeoxy Terminator Cycle
Sequencing Kit (Perkin Elmer, Foster City, CA).
Transfection of Daudi cells
Daudi cells were harvested in exponential growth phase, washed
twice, and suspended to 1.35 × 107/mL in OPTI-MEM.
To 800 µL of cell suspension in a 0.4-cm electroporation cuvette, 20 µg of pGL3-5' flanking region (or deletions) of PLSCR1 and 20 µg of pSV--galactosidase (Promega) were added, and the mixture was
incubated for 10 minutes on ice. Electroporation was performed at 380 V
and 500 µF using a Bio-Rad Gene Pulser II (Bio-Rad Laboratories,
Hercules, CA). Following incubation for 10 minutes at 37°C, the
cells were plated in 10 mL RPMI-1640 complete medium onto tissue
culture plates and cultured for 24 hours. Cells were then cultured for
an additional 18 hours in the presence or absence of 1000 IU/mL of
IFN-2a, and harvested for luciferase and -galactosidase assay.
Luciferase and -galactosidase assay
Luciferase activity was measured using a Luciferase Assay Kit
(Promega). In brief, Daudi cells were harvested, washed with PBS, and
lysed for 15 minutes with reporter lysis buffer. Cell lysates were
vortexed for 15 seconds and centrifuged at 12,000g for 2 minutes at 4°C. In a 96-well plate, 20-µL aliquots of lysate (18 µg protein) were mixed with 100 µL of luciferase assay buffer by
automated injection using a MicroLumatPlus microplate
luminometer (EG&G Berthold, Gaithersburg, MD), and luminescence was
measured for a period of 30 seconds. -Galactosidase activity was
determined with o-nitrophenyl--D-galactopyranoside
(OPNG) as a substrate. A 100-µL aliquot of cell lysate (90 µg
protein) was incubated with 100 µL of 4.4 mM ONPG for 1 hour at
37°C, and absorbance was read at 420 nm. Luciferase activity was
expressed in arbitrary light units and corrected for transfection
efficiency of -galactosidase.
Evaluation of cell surface-exposed phosphatidylserine in
adherent cells
The cell surface exposure of phosphatidylserine resulting from
treatment with IFN induction and ionophore treatments of adherent cell
lines HT1080 and human umbilical vein endothelial cells was evaluated
by expression of membrane catalytic function in the prothrombinase
enzyme reaction. Cells were grown to about 80% confluence in a 48-well
culture plate and induced overnight (18 hours) with either 0 or 1000 IU/mL IFN-2a. After 3 washes, cells were incubated at 37°C in
the presence of either 5 µM (HT1080) or 10 µM (human umbilical vein
endothelial cells) A23187 in Hanks' balanced salt solution (HBSS)
containing 2 mM Ca++, 0.8 mM Mg++, and 0.1%
BSA for the time periods indicated. Controls omitting A23187 received
identical 1% (final volume) solvent DMSO. During the last 2 minutes of
treatment with A23187, the prothrombinase reaction was initiated by
addition of factor Va (2 nM), factor Xa (1 nM), and prothrombin (1.4 µM). Thrombin generation was terminated by dilution of cell
supernatants into HBSS containing 0.1% BSA and 20 mM EDTA, and samples
were stored on ice. Aliquots were transferred to a 96-well plate, and
thrombin generated was assayed in HBSS containing 0.1% BSA in the
presence of 150 µM chromogenic substrate CBS 34.47 by monitoring
time-dependent changes in absorbance at 405 nm using a
Thermomax plate reader (Molecular Devices, Sunnyvale, CA).
Flow cytometry
Interferon- and A23187-induced cell surface exposure of
phosphatidylserine was evaluated in the suspension cell lines Daudi and
Raji using flow cytometric detection of bound factor Va (light chain)
as previously described.8 Following 18 hours of induction with either 0 or 1000 IU/mL IFN-2a, cells were washed once with RPMI
and suspended (3 × 106 cells/mL) in RPMI containing
0.1% BSA, 4 mM Ca++. After 2 minutes at 37°C, A23187
(0 or 1 µM) was added. At each time point, the reaction was stopped
by addition of 10 mM EGTA, and cells incubated with bovine factor Va
(10 µg/mL, 15 minutes at room temperature) and bound factor Va were
detected with mab FITC-V237 specific for the light chain.13
Cells staining positive for bound factor Va were analyzed by flow
cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes,
NJ). Data were expressed as percentage of gated
factor Va-positive cells in the total cell population.
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Results |
Previous screening by high-density oligonucleotide microarrays
provided evidence of an induction of PLSCR1 mRNA in HT1080 cells 6 hours after exposure to IFN-, , or .5 These
findings were extended by demonstrating the time-dependent induction of both PLSCR1 mRNA and protein by IFN-2a in Northern and Western blots
(Figures 1
and 2, respectively). Increased PLSCR1 mRNA was detected by 3 hours
after IFN-2a (1000 IU/mL) addition, with protein expression
increasing to approximately 10-fold above basal levels at 18 hours.
Peak expression of PLSCR1 mRNA was observed at 6 hours, the same length
of IFN treatment as in the microarray analysis.5 By
contrast to this response observed in the IFN-responsive HT1080 cells,
treatment with IFN-2a had no effect on PLSCR1 expression in mutant
U3A cells, an HT1080 derivative cell line deficient in STAT1
transcription factor required for signaling through
IFN-receptors.14
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| Fig 1.
Induction of PLSCR1 mRNA by IFN-2a.
Northern blotting of human PLSCR1 in HT1080 cells (panel A) and in
STAT-defective U3A cells (panel B) was performed using full-length cDNA
for PLSCR1. Times indicate hours after addition of 1000 IU/mL IFN-2a
(see "Materials and methods"). Also shown are results obtained
when same blots were probed with control cDNAs for GAPDH and
-actin.
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| Fig 2.
Expression of PLSCR1 in HT1080 cells.
Western blotting for PLSCR1 expressed in HT1080 (left panel) and U3A
(right panel) cells after treatment with 1000 IU/mL IFN-2a under
conditions of Figure 1 was performed with mab 4D2 (see "Materials
and methods"). The IFN-induced increase in PLSCR1 antigen detected
in HT1080 (normalized to t = 0) was 1.5-fold (t = 3 hours),
4.3-fold (t = 6 hours), 8.3-fold (t = 18 hours), and 6.8-fold
(t = 24 hours). In U3A, the increase was 1.4-fold (t = 3 hours),
1.3-fold (t = 6 hours), 1.3-fold (t = 18 hours), and 1.2-fold
(t = 24 hours). Data of single experiment, representative of 3 so
performed.
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The IFN-2a dependence of PLSCR1 expression observed in HT1080 cells
was confirmed in a variety of other transformed cell lines as well as
in cultures of human umbilical vein endothelial cells and
nontransformed peripheral blood mononuclear cells isolated from whole
blood (Figure 3 and data not shown). In all
cases, incubation with IFN-2a caused a marked increase in PLSCR1
protein expression, ranging to as high as 10-fold above basal levels in the Raji and Daudi cell lines. These data indicate that the
PLSCR1 gene is highly up-regulated by IFN-2a treatment in a
variety of normal and transformed cell types.
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| Fig 3.
Induction of PLSCR1 by IFN-2a in various human cell
lines.
Western blotting for PLSCR1 was performed in cells indicated after 18 hours of treatment with 0 () or 1000 IU/mL (+)
IFN-2a. HUVEC denotes human umbilical vein endothelial
cells. The IFN-induced increase in PLSCR1 antigen detected was 2-fold
(Jurkat), 3-fold (HUVEC, HELA), and 10-fold (Daudi, Raji). Data of
single experiment, representative of at least 3 similar experiments so
performed on each cell line.
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After IFN treatment, newly synthesized PLSCR1 was detected in the
plasma membrane where it appeared to concentrate in membrane protrusions. In addition to plasma membrane, PLSCR1 antigen also appeared to be distributed in a variety of other intracellular membranous structures, suggestive of Golgi and endoplasmic reticulum (Figure 4).
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| Fig 4.
Fluorescence microscopy.
HT1080 cells were incubated with 1000 IU/mL IFN-2a for various times
indicated. After fixation and permeabilization, PLSCR1 antigen (green
fluorescence) was detected using mab 4D2 (see "Materials and
methods"). Cell nuclei are counterstained with propidium iodide (red
fluorescence). Control IgG refers to IFN-induced cells that were
identically treated and stained with IgG1 murine antibody to irrelevant
antigen (complement C9) substituting for mab 4D2. Data representative
of 2 independent experiments so performed. White bar indicates 50 µ scale.
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Inspection of PLSCR1 genomic sequence revealed 3 potential
IFN-regulated sites within the first 4 kb of 5' flanking
sequence; a potential binding site for IRF-2 at
(3815)gaaaagaGAATcc(3800); potential binding sites for
ISGF3 at (2733)acaaaaaGAAAgc(2721) and at
(2519)aaaaacaGAAAcc(2497), and a single consensus
interferon-stimulated response element (ISRE) in the untranslated exon
1 at (+21)gggaaaagGAAAccg(+35) (Figure 5).
To identify which of these 4 putative regulatory sites actually
contributed to the observed IFN-inducible expression of PLSCR1,
luciferase reporter constructs incorporating 5' untranslated PLSCR1 gene sequence spanning 1 or more of the
putative sites were expressed in Daudi cells, and the response of the
transfected cells to IFN-2a was determined. As shown in Figure 5,
these experiments revealed that the IFN-inducible expression of
PLSCR1 appears to be controlled by the single ISRE that is
located in exon 1. The close proximity of this ISRE to the PLSCR1
transcriptional start site may account for the observed potency of
IFN-2a in inducing PLSCR1 expression.
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| Fig 5.
Identity of ISRE in PLSCR1 genomic sequence.
Four putative ISRE-like elements (filled boxes) located between
4120 bp and +60 bp of the 5' flanking region and first
untranslated exon of PLSCR1 gene are depicted in linear map
(top): #4 = (3815)gaaaagaGAATcc(3800);
#3 = (2733)acaaaaaGAAAgc(2721);
#2 = (2519aaaaacaGAAAcc(2497);
#1 = (+21)gggaaaagGAAAccg(+35). Arrow denotes transcription
initiation site. Sequence spanning these various putative ISRE-like
elements were selectively deleted by PCR and the truncated PLSCR1 DNA
cloned into pGL3-luciferase reporter vector as described in
"Materials and methods." Daudi cells were then cotransfected with
-galactosidase-pSV (as transfection efficiency control) and these
PLSCR1-pGL3-luciferase plasmids containing the following insertions of
PLSCR1 5' genomic DNA: 4120 bp to +60 bp (spanning
#1-4); 3307 bp to +60 bp (spanning #1-3); 2277 bp
to +60 bp (spanning #1 only); 4120bp to +18 bp (spanning #2-4);
and pGL3 vector without insert (vector). After 24 hours of
transfection, either 0 (solid bars) or 1000 IU/mL (open bars) IFN-2a
was added to the cell cultures, and 18 hours later, the cells were
harvested for measurement of luciferase and -galactosidase
activities (see "Materials and methods"). Bar graph reports ratio
of luciferase/-galactosidase activities measured at 18 hours. Error
bars denote mean ± SEM (n = 3). Data of single experiment,
representative of 3 experiments so performed. The average IFN-induced
increase (mean ± SD) obtained for each reporter construct from the
combined data of all 3 experiments was 3.9 ± 0.4
(insert spanning #1-4); 5.3 ± 1.0 (insert spanning #1-3);
5.2 ± 0.8 (insert spanning #1); 0.8 ± 0.1 (insert spanning
#2-4); 0.8 ± 0.2 (vector control).
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In reconstituted proteoliposomes, PLSCR1 has been shown to mediate
accelerated transbilayer migration of membrane phospholipids in the
presence of Ca++ or under acidic
conditions.6,7,9,15 Furthermore, the level of expression of
this protein was generally found to correlate with the extent to which
phosphatidylserine was exposed at the cell surface following calcium
ionophore treatment, suggesting that PLSCR1 participates in the
remodeling of plasma membrane phospholipids in activated platelets and
injured or apoptotic cells exposed to increased intracellular Ca++
concentration.8 A potential influence of PLSCR1 on
either cell proliferation or cell clearance in vivo was also suggested
by the observation of altered transcription including alternative splicingof a murine PLSCR1 orthologue in leukemogenic versus nonleukemogenic cell clones.16,17 We therefore considered
whether the marked up-regulation of PLSCR1 induced by IFN-2a is also accompanied by changes in the plasma membrane that might increase the
likelihood of phosphatidylserine becoming exposed at the cell surface.
Despite the presumed activity of PLSCR1 in mediating accelerated
transbilayer movement of plasma membrane phospholipids leading to
transfer of phosphatidylserine to the outer leaflet, we were not able
to detect any change in the IFN-2a-treated cells indicative of
increased surface exposure of plasma membrane phosphatidylserine (Figures 6
and 7).
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| Fig 6.
Prothrombinase assay for cell surface phosphatidylserine
exposed in IFN-2a-induced cells.
Cells were incubated 18 hours with either 0 (open symbols) or 1000 IU/mL (closed symbols) IFN-2a. After induction with IFN-2a, cells
were challenged with calcium ionophore (open and closed squares) and
prothrombinase activity measured as a function of time after addition
of A23187 (abscissa). Open and closed circles denote results for
identically matched cells omitting A23187. Upper panel shows results
for cell line HT1080 receiving 5 µM A23187; lower panel shows results
for human umbilical vein endothelial cells receiving 10 µM A23187.
Results (mean ± SD) are plotted from a single experiment
representative of similar experiments performed at least 3 times with
each cell type.
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| Fig 7.
Flow cytometry.
The suspension cell line Daudi (upper panel) or Raji (lower panel) was
incubated 18 hours with either 0 (open squares) or 1000 IU/mL (closed
circles) IFN-2a and then analyzed for cell surface-exposed
phosphatidylserine, as detected by the binding of coagulation
factor Va (FVa; see "Materials and methods"). Ordinate
represents percent of cells analyzed by flow cytometry detected as
positive for bound FVa. Error bars represent mean and range of combined
results of 2 independent experiments. Similar data were obtained for
Jurkat cells (not shown).
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Discussion |
These experiments demonstrate that the PLSCR1 gene is a
member of the IFN-stimulated gene family requiring JAK/STAT signaling for optimal expression. The locus of the controlling ISRE in
the untranslated first exon of the PLSCR1 gene represents a putative binding site for the ISGF3 transcription factor complex. Whereas most
known ISREs map to flanking sequence that is 5' to the
transcriptional start site, the location of an active ISRE in
untranslated exonic sequence has also previously been described for the
p202 gene, another IFN-stimulated gene regulated through
ISGF3.18 As has been noted in other IFN-stimulated genes,
the marked effect of IFN-2a in up-regulating PLSCR1 expression is
consistent with the relatively close proximity of this single active
ISRE to the transcriptional start site.
Certain of the IFN-stimulated genes regulated through ISREs
are thought to be involved in the apoptotic, antiproliferative, and
tumor suppressive activities of IFN-, although the precise roles of
the downstream effector genes actually responsible for these activities
remain to be resolved.19-21 Induction of apoptosis of
malignant cells or virus-infected cells by IFNs, and clearance of these
apoptotic cells by the reticuloendothelial system, are widely assumed
to underlie the therapeutic response to IFN treatment. In light of the
putative role of the PLSCR1 gene product in catalyzing movement of
phospholipids between plasma membrane leaflets, it is of particular
interest that one of the most prominent changes observed in
apoptotic cells is a remodeling of the topology of plasma membrane
phospholipids, with surface exposure of phosphatidylserine and other
aminophospholipids that are normally sequestered to the inner leaflet.
Such cell surface exposure of phosphatidylserine has been implicated in
promoting clearance of injured or apoptotic cells by the
reticuloendothelial system.10,11,22
Phospholipid scramblase is an endofacial-oriented plasma
membrane protein that has been proposed to contribute to accelerated movement of phospholipids between plasma membrane leaflets in activated
platelets as well as in injured and apoptotic cells that are exposed to
local elevations in Ca++ concentration or to acidification
affecting the inner plasma membrane leaflet.9-12 This
activity of the PLSCR1 gene product in promoting Ca++ and
pH-dependent movement of phospholipids between membrane leaflets was
demonstrated in reconstituted proteoliposomes containing this protein,
and the level of cellular expression of PLSCR1 was previously found in
general to correlate with the observed extent of transfer of
phosphatidylserine to the cell surface in response to induced elevations of cytoplasmic Ca++ concentration.
Nevertheless, the exact role of this protein in promoting
transbilayer movement of phosphatidylserine and other plasma membrane
phospholipids and the actual mechanism of activation of the
phospholipid scramblase pathway in situ remains to be clarified. As
noted above, induction of PLSCR1 by IFN-2a leads to a marked
increase in concentration of phospholipid scramblase that is expressed
in the plasma membrane of the IFN-treated cells, but we were
unsuccessful in detecting either a corresponding increase in
surface-exposed phosphatidylserine or increased sensitivity of the
plasma membranes of these cells to subsequent treatment with calcium
ionophore. These data suggest that the mobilization of
phosphatidylserine to the cell surface cannot simply be attributed to
the level of expression of the PLSCR1 gene product as was previously
assumed, but is likely to require additional factors, including
potentially another protein, that either acts directly on the plasma
membrane or that interacts with PLSCR1 to accelerate transbilayer
movement of phospholipids in the plasma membrane. Alternatively, the
endogenous level of PLSCR1 expressed in the plasma membrane of these
cells before IFN treatment may itself be sufficient to mediate maximal
response to calcium ionophore under the conditions of these
experiments, masking more subtle changes in phospholipid trafficking
between plasma membrane leaflets arising from the IFN-induced increase in plasma membrane concentration of the protein. It also remains to be
determined whether the marked increase in PLSCR1 expression induced by
IFN promotes remodeling of plasma membrane phospholipids or cell
clearance in vivo, under physiologic conditions relevant to tumor
growth and viral infection.
The conclusions of the present experiments are consistent with recent
observations relating to the possibility that mutation affecting PLSCR1
was responsible for the cellular defect underlying Scott syndrome, a
rare inherited bleeding disorder characterized by reduced mobilization
of phosphatidylserine to the surface of activated platelets and other
blood cells.23,24 When PLSCR1 expressed in Scott blood
cells was analyzed, the deduced amino acid sequence and levels of
expressed mRNA and protein were normal.25 Furthermore,
phospholipid scramblase derived from Scott cell membranes showed
apparently normal function once extracted in detergent and
reconstituted in proteoliposomes.26 Thus, despite the
aberrant properties of the plasma membranes of the Scott syndrome cells suggesting an inherited defect or deficiency affecting the plasma membrane phospholipid scramblase pathway, no abnormality of PLSCR1 per
se was detected. This raised the likelihood that another gene is
aberrantly expressed or mutated in patients with Scott syndrome, affecting either another as yet unidentified protein with such activity, or alternatively, a regulatory cofactor required for normal
PLSCR1 function in situ.
The IFNs have been used for more then 10 years in the treatment of
various diseases in humans, including chronic viral syndromes and
various malignant and nonmalignant cell proliferative
disorders.1 The clinical outcome of IFN treatment is
variable. The IFNs induce remission in some patients who have good
response to these cytokines and can lead to improvement in these
patients. On the other hand, the same IFN therapy results in no benefit
at all in some patients who have poor response to IFNs and such
treatment can result in severe side effects.27,28 The
mechanisms underlying the antitumor or antivirus effects of IFNs and
the reason for resistance to IFN therapy in some patients are poorly
understood. The discovery of a new IFN-stimulated gene with induced
mRNA and protein that can be readily detected in IFN-responsive cells
may provide a new tool for quantifying or predicting response to IFN
therapy, and promises to shed new insight into the molecular and
cellular events that underlie the diverse biologic activities of these cytokines.
| |
Acknowledgments |
The authors acknowledge the gift of cell line U3A from Dr George R. Stark (The Cleveland Clinic Foundation) and antibody V237 from Dr
Charles T. Esmon (The Oklahoma Medical Research Foundation). The
assistance of Dr Malcolm Wood (The Scripps Research Institute) with
confocal microscopy is gratefully acknowledged as is the superb
technical assistance of Lilin Li, Hongfan Peng, and Yolanda Montejano.
| |
Footnotes |
Submitted September 27, 1999; accepted December 21, 1999.
Supported by grants HL36946 (P.J.S.), HL61200 (T.W.), and
HL63819 (P.J.S.) from the Heart, Lung, and Blood Institute, and by
grant CA44059 (R.H.S.) from the National Cancer Institute, National
Institutes of Health, and by the Stein Endowment Fund.
Reprints: Peter J. Sims, Departments of Molecular and
Experimental Medicine and Department of Vascular Biology, The Scripps
Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037;
e-mail: psims{at}scripps.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD.
How cells respond to interferons.
Annu Rev Biochem.
1998;67:227[Medline]
[Order article via Infotrieve].
2.
Doly J, Civas A, Navarro S, Uze G.
Type I interferons: expression and signalization.
Cell Mol Life Sci.
1998;54:1109[Medline]
[Order article via Infotrieve].
3.
Darnell JEJ, Kerr IM, Stark GR.
Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
Science.
1994;264:1415[Abstract/Free Full Text].
4.
Duhe RJ, Farrar WL.
Structural and mechanistic aspects of Janus kinases: how the two-faced god wields a double-edged sword.
J Interferon Cytokine Res.
1998;18:1[Medline]
[Order article via Infotrieve].
5.
Der SD, Zhou AM, Williams BR, Silverman RH.
Identification of genes differentially regulated by interferon , , or gamma using oligonucleotide arrays.
Proc Natl Acad Sci U S A.
1998;95:15,623[Abstract/Free Full Text].
6.
Bassé F, Stout JG, Sims PJ, Wiedmer T.
Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid.
J Biol Chem.
1996;271:17,205[Abstract/Free Full Text].
7.
Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ.
Molecular cloning of human plasma membrane phospholipid scramblasea protein mediating transbilayer movement of plasma membrane phospholipids.
J Biol Chem.
1997;272:18,240[Abstract/Free Full Text].
8.
Zhao J, Zhou Q, Wiedmer T, Sims PJ.
Level of expression of phospholipid scramblase regulates induced movement of phosphatidylserine to the cell surface.
J Biol Chem.
1998;273:6603[Abstract/Free Full Text].
9.
Stout JG, Zhou QS, Wiedmer T, Sims PJ.
Change in conformation of plasma membrane phospholipid scramblase induced by occupancy of its Ca2+ binding site.
Biochemistry.
1998;37:14,860[Medline]
[Order article via Infotrieve].
10.
Verhoven B, Krahling S, Schlegel RA, Williamson P.
Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes.
Cell Death Differ.
1999;6:262[Medline]
[Order article via Infotrieve].
11.
Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM.
The role of phosphatidylserine in recognition of apoptotic cells by phagocytes.
Cell Death Differ.
1998;5:551[Medline]
[Order article via Infotrieve].
12.
Bevers EM, Comfurius P, Dekkers DWC, Harmsma M, Zwaal RFA.
Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance.
Biol Chem.
1998;379:973.
13.
Sims PJ, Faioni EM, Wiedmer T, Shattil SJ.
Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity.
J Biol Chem.
1988;263:18,205[Abstract/Free Full Text].
14.
McKendry R, John J, Flavell D, Muller M, Kerr IM, Stark GR.
High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons.
Proc Natl Acad Sci U S A.
1991;88:11,455[Abstract/Free Full Text].
15.
Zhou Q, Sims PJ, Wiedmer T.
Identity of a conserved motif in phospholipid scramblase that is required for Ca2+-accelerated transbilayer movement of membrane phospholipids.
Biochemistry.
1998;37:2356[Medline]
[Order article via Infotrieve].
16.
Kasukabe T, Okabe-Kado J, Honma Y.
TRA1, a novel mRNA highly expressed in leukemogenic mouse monocytic sublines but not in nonleukemogenic sublines.
Blood.
1997;89:2975[Abstract/Free Full Text].
17.
Kasukabe T, Kobayashi H, Kaneko Y, Okabe-Kado J, Honma Y.
Identity of human normal counterpart (MmTRA1b) of mouse leukemogenesis-associated gene (MmTRA1a) product as plasma membrane phospholipid scramblase and chromosome mapping of the human MmTRA1b phospholipid scramblase gene.
Biochem Biophys Res Commun.
1998;249:449[Medline]
[Order article via Infotrieve].
18.
Samanta H, Engel DA, Chao HM, Thakur A, Garcia-Blanco MA, Lengyel P.
Interferons as gene activators. Cloning of the 5' terminus and the control segment of an interferon activated gene.
J Biol Chem.
1986;261:11,849[Abstract/Free Full Text].
19.
Tanaka N, Ishihara M, Kitagawa M, et al.
Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1.
Cell.
1994;77:829[Medline]
[Order article via Infotrieve].
20.
Tamura T, Ishihara M, Lamphier MS, et al.
An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes.
Nature.
1995;376:596[Medline]
[Order article via Infotrieve].
21.
Escalante CR, Yie J, Thanos D, Aggarwal AK.
Structure of IRF-1 with bound DNA reveals determinants of interferon regulation.
Nature.
1998;391:103[Medline]
[Order article via Infotrieve].
22.
Diaz C, Schroit AJ.
Role of translocases in the generation of phosphatidylserine asymmetry.
J Membr Biol.
1996;151:1[Medline]
[Order article via Infotrieve].
23.
Weiss HJ.
Scott syndrome: a disorder of platelet coagulant activity.
Semin Hematol.
1994;31:312[Medline]
[Order article via Infotrieve].
24.
Satta N, Toti F, Fressinaud E, Meyer D, Freyssinet J-M.
Scott syndrome: an inherited defect of the procoagulant activity of platelets.
Platelets.
1997;8:117.
25.
Zhou QS, Sims PJ, Wiedmer T.
Expression of proteins controlling transbilayer movement of plasma membrane phospholipids in the B lymphocytes from a patient with Scott syndrome.
Blood.
1998;92:1707[Abstract/Free Full Text].
26.
Stout JG, Bassé F, Luhm RA, Weiss HJ, Wiedmer T, Sims PJ.
Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.
J Clin Invest.
1997;99:2232[Medline]
[Order article via Infotrieve].
27.
Williams CD, Linch DC.
Interferon alfa-2a.
Br J Hosp Med.
1997;57:436[Medline]
[Order article via Infotrieve].
28.
Grander D, Einhorn S.
Interferon and malignant diseasehow does it work and why doesn't it always?
Acta Oncol.
1998;37:331[Medline]
[Order article via Infotrieve].
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Abstract
Introduction