From the Centre for Thrombosis and Vascular
Research, The University of New South Wales, Sydney NSW 2052, Australia
and the § Scripps Research Institute, La Jolla, California
92037
Received for publication, October 10, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis of smooth muscle cells (SMC)
in atherosclerotic vessels can destabilize the atheromatus plaque and
result in rupture, thrombosis, and sudden death. In efforts to
understand the molecular processes regulating apoptosis in this cell
type, we have defined a novel mechanism involving the ubiquitously
expressed transcription factor Sp1. Subtypes of SMC expressing abundant
levels of Sp1 produce the death agonist, Fas ligand (FasL) and undergo
greater spontaneous apoptosis. Sp1 activates the FasL promoter via a
distinct nucleotide recognition element whose integrity is crucial for inducible expression. Inducible FasL promoter activation is also inhibited by a dominant-negative form of Sp1. Increased SMC apoptosis is preceded by Sp1 phosphorylation, increased FasL transcription, and
the autocrine/paracrine engagement of FasL with its cell-surface receptor, Fas. Inducible FasL transcription and apoptosis are blocked
by dominant-negative protein kinase C- Apoptosis is a genetically regulated "programmed" form of cell
death and is characterized by a number of specific biochemical and
morphological changes, including nuclear chromatin condensation, cytoplasmic condensation, membrane blebbing, and internucleosomal fragmentation of DNA (1, 2). Fas/APO-1 (or CD95) is a 45-kDa cell
surface glycoprotein that belongs to the tumor necrosis factor receptor
superfamily and mediates apoptosis in various normal and transformed
cell types. Upon the engagement of Fas by Fas ligand
(FasL),1 a highly conserved,
ubiquitously expressed 40-kDa glycoprotein, the apoptotic cysteine
protease caspase-8/FLICE is recruited to the receptor via FADD and
activated by proteolysis (3). Caspase-3/CPP32, which is expressed in
cells as the inactive 32-kDa form is, in turn, cleaved by
caspase-8/FLICE to produce two mature subunits (17 and 12 kDa). Active
caspase-3/CPP32 cleaves nuclear mitotic apparatus protein and mediates
DNA fragmentation, chromatin condensation, and the formation of
apoptotic bodies (4).
FasL (5) and Fas (6, 7) are both expressed in arterial tissue,
including the human atherosclerotic plaque. Immunohistochemical analysis revealed FasL expression in 34 of 34 carotid atherosclerotic plaques examined, with virtually all FasL positive-staining associated with intimal smooth muscle cells (SMCs) and little staining apparent in
normal arterial tissue (5). Fas is also highly expressed in intimal
SMCs of the plaque (6, 7). FasL/Fas expression and apoptosis (8-10) in
normal artery and plaque has prompted speculation on the roles of
these molecular mediators in vascular cells. Apoptosis in undiseased
tissue may inhibit arterial thickening by limiting cell proliferation
and accumulation in the intima (6). In atherosclerotic tissue,
apoptosis particularly of collagen-producing SMCs may substantially
weaken the plaque causing it to rupture, initiate thrombosis, and
trigger acute coronary syndromes (11-12). Overexpression of FasL in
balloon-injured rat carotid arteries devoid of endothelium-induced
apoptosis in medial SMCs and inhibited intimal hyperplasia (13, 14).
However, recent evidence in a rabbit model suggests that FasL may
promote rather than retard atherogenesis. FasL overexpression in
nondenuded arteries of hypercholesterolemic animals stimulated lesion
formation in these animals via increased cellularity (15). These
observations may be due to differences in artery and lesion cellular
composition or cholesterol feeding between the two animal models.
Despite clear evidence for FasL and Fas expression in SMCs of the
artery wall, the molecular mechanisms mediating FasL production in
vascular cells are presently not known. The promoter region of the FasL
gene has recently been cloned and found to contain binding sites for a
number of transcription factors including NF- The discovery and functional characterization of Sp1 as a GC-rich
binding nuclear protein has provided a useful paradigm to our
understanding of the regulation of transcriptional activation in
eukaryotic cells (21, 22). Sp1 is a broadly expressed nuclear protein
of ~100 kDa and contains three Kruppel-like zinc fingers that contact
DNA (21, 22). A nucleotide recognition element for Sp1 is located in
the FasL promoter at position WKY12-22 and WKY3M-22 cells are well established subtypes of vascular
smooth muscle cells that are phenotypically distinct (24, 25). WKY12-22
cells have a cobblestone morphology in culture, proliferate in
plasma-derived serum (which lacks vital growth factors), and
spontaneously overexpress mRNA for platelet-derived growth factor
(PDGF) B-chain, elastin, and osteopontin (24, 25). In contrast,
WKY3M-22 cells are typically spindle-shaped and do not express PDGF-B,
elastin, or osteopontin mRNA, nor do they grow in plasma-derived
serum. Both cell subtypes are phenotypically stable in culture and can
be passaged indefinitely. Therefore, WKY12-22 and WKY3M22 cells
represent important cells with which to delineate the molecular basis
for differences in SMC phenotype and gene expression.
We recently reported that Sp1 is spontaneously expressed at
greater levels in WKY12-22 cells than WKY3M-22 cells
and that as a consequence, Sp1-dependent genes, such as
PDGF-B, are overexpressed in WKY12-22 cells compared with
its sister cell subtype (26). These observations provided important
insight into the transcriptional basis for differential gene
expression. Here we explored the regulatory role of Sp1 in inducible
FasL expression and apoptosis in two phenotypically distinct SMC subtypes.
Transfections and Luciferase Assays--
SMCs were maintained
in Waymouth's medium (Life Technologies, Inc.), pH 7.4, containing
10% fetal bovine serum at 37 °C in a humidified atmosphere of 5%
CO2. Transient transfections were performed with cells at
60% confluence, and the indicated constructs together with 2 µg of
the internal control vector, pRL-TK, using FuGENE6 transfection agent
(Roche Molecular Biochemicals). After 24 h, the transfected cells
were incubated with or without CAM (1 µg/ml), and luciferase activity
was quantified using the Dual Luciferase Assay System (Promega).
Firefly luciferase activity was normalized to Renilla data
generated from pRL-TK.
Plasmid Constructs--
Various sized fragments of the FasL
promoter ( Quantitative Assessment of DNA Fragmentation--
SMCs were
grown in 96-well plates to 80% confluency in 100 µl of growth
medium. Where indicated, the cells were incubated with Fas-Fc (R&D
Systems) and/or IgG Fc (R&D Systems) (50 µg/ml, final concentration)
for 1 h prior to the addition of CAM. After 24-h exposure to CAM,
apoptosis was quantitated using the Cell Death Detection
ELISAPlus (Roche Molecular Biochemicals). This assay, which
measures cytoplasmic histone-associated internucleosomal DNA
fragmentation, has been used previously to quantitate inducible
apoptosis in cultured cells (27-29). Briefly, the cells were washed
gently in PBS and incubated with shaking in lysis buffer for 30 min at
22 °C. Lysates were transferred into Eppendorf tubes and spun at
14,000 rpm for 30 s. Twenty µl of the supernatant was used in
the ELISA, which was performed in accordance with the manufacturer's
instructions and normalized to total cell number measured using a
Coulter counter. Results are expressed as total internucleosomal DNA
fragmentation as a proportion of the cell population.
Annexin V Staining/FACS Analysis--
SMCs were washed twice
with ice-cold phosphate-buffered saline, pH 7.4 and resuspended in 1×
binding buffer (10 mM HEPES, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2) at a concentration of
1 × 106 cells/ml. One hundred microliters of the
suspension was transferred to 5-ml flat bottomed tubes where 5 µl of
annexin V-fluorescein isothiocyanate and 10 µl of propidium iodide
(50 µg/ml stock in PBS) was added. The cells were gently vortexed and
incubated in the dark at 22 °C for 15 min. Four hundred microliters
of binding buffer was added to each tube, and annexin V staining was
analyzed by flow cytometry within 1 h. Results are expressed as
annexin V staining as a percentage of the total cell population.
Propidium Iodide Nuclear Staining--
SMCs were grown in
chamber slides (80% confluent) and incubated with CAM (1 µg/ml) for
24 h. The cells were washed in PBS, pH 7.4, and fixed with
methanol/acetone (80:20) for 10 min at 22 °C. Propidium iodide (50 µM) was added to each well and incubated for a maximum of
5 min followed by a second wash with PBS. Cells undergoing apoptosis
were visualized by confocal microscopy.
Nuclear Extract Preparation--
SMCs treated with CAM for
various times were washed and scraped in 10 ml of PBS and transferred
to precooled centrifuge tubes. Samples were spun at 1300 rpm for 15 min
at 4 °C. The pellet was resuspended in 100 µl (for two 100-mm
dishes) of solution A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl) and placed on
ice for 5 min. Samples were spun at 14,000 rpm for 40 s. The
pellet was resuspended in 20 µl of solution C (20 mM
Hepes, pH 7.9, 1.5 mM MgCl2, 420 mM
NaCl, 0.2 mM EDTA) and mixed gently for 20 min at 4 °C.
The supernatant was transferred to precooled Eppendorf tubes containing
20 µl of solution D (20 mM Hepes, pH 7.9, 1.5 mM KCl, 0.2 mM EDTA, 20% glycerol) and stored
at Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts (6-10 µg) were incubated with 32P-labeled
double-stranded oligonucleotide (150,000 cpm, 40 fmol) in 20 µl
containing 10 mM Tris-HCl, pH 8.0, 50 mM
MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 5% glycerol, 1 µg of salmon sperm DNA, 5% sucrose,
1 µg of poly(dI-dC) and 1 mM phenylmethylsulfonyl
fluoride. The mixture was incubated for 35 min at 22 °C. In
supershift experiments, nuclear extract was incubated with 2 µg of
antibody prior to addition of the probe. Samples were resolved by 8%
nondenaturing polyacrylamide gel electrophoresis, and binding complexes
were visualized by autoradiography at Western Blot for Sp1--
Fifteen micrograms of nuclear extract
was resolved by 8% SDS-polyacrylamide gel electrophoresis and then
transferred onto Immobilon-P transfer membranes (Millipore). The
membranes were blocked overnight at 4 °C in PBS containing 5% skim
milk and 0.05% Tween 20. Sp1 was detected with Sp1 polyclonal
antibodies (1:1000, Santa Cruz Biotechnology) and subsequent
chemiluminescent visualization.
Sp1 Dephosphorylation Analysis by Western Blotting and
EMSA--
Nuclear extracts (10-15 µg) were incubated with or
without 5 units of calf intestinal alkaline phosphatase (CIP, NEB) for 1 h at 37 °C in a total volume of 20 µl. The reaction was
quenched by the addition of loading dye prior to 8% SDS-polyacrylamide gel electrophoresis and Western blot analysis for Sp1. In EMSA, 8 µg
of nuclear extract was incubated with 5 milliunit of CIP (final
concentration determined by CIP titration experiments with 32P-labeled FasL Oligo) at 33 °C for 5 min and then on
ice for 15 min. The reaction was stopped by the addition of phosphatase
inhibitors at a final concentration of 10 nM LiNaF, 10 nM sodium vanadate, 10 nM potassium
pyrophosphate, and 5 nM sodium phosphate. EMSA was
performed as described above. CIP treatment prior to EMSA has
previously been used for the assessment of Sp1 phosphorylation in
nuclear extracts (30, 31).
RT-PCR--
Five micrograms of total RNA isolated using
TRIzol reagent (Life Technologies, Inc.) were treated with DNase I, and
cDNA was generated using Superscript II reverse transcriptase (Life
Technologies, Inc.) with random primers according to the
manufacturer's instructions. Sequences of the primers for FasL,
Fas, and
PCR was performed in a total volume of 50 µl containing 2 mM MgCl2, 2 mM dNTPs, 2.5 units of
Taq DNA polymerase (Sigma), 5 µl of cDNA, and either
100 pmol of Fas primers, 100 pmol of FasL primers, or 20 pmol of
Northern Blot Analysis--
Fifteen µg of total RNA
isolated using TRIzol reagent (Life Technologies, Inc.) was loaded onto
a 1% formaldehyde/agarose gel and resolved by electrophoresis.
Northern blot was performed as previously described (32). FasL and
glyceraldehyde-3-phosphate dehydrogenase cDNA amplified by PCR were
used as probes. The cDNA was labeled by nick translation (Roche
Molecular Biochemicals).
Sp1 Is Proapoptotic--
To begin investigating a possible
mechanistic role for Sp1 in programmed cell death, we compared
apoptosis in two well established SMC subtypes isolated originally from
the arteries of pup (2-week-old) and adult (3-month-old) rats (24, 25).
Pup SMCs (WKY12-22 cells) are phenotypically distinct from their adult
counterparts (WKY3M-22 cells) and express abundant levels of Sp1 (26).
We found that cytoplasmic histone-associated internucleosomal DNA fragmentation (27-29) is greater in WKY12-22 cells than WKY3M-22 cells
(Fig. 1A, left),
indicating higher levels of spontaneous apoptosis in the former cell
subtype and providing further evidence that these SMC subtypes are
phenotypically distinct. These observations were confirmed by
annexin V-fluorescein isothiocyanate staining upon
fluorescence-activated cell sorting, indicating greater disrupted membrane symmetry exposing phosphatidylserine to the external environment (Fig. 1A, right). Similar findings
were obtained qualitatively by DNA laddering on ethidium
bromide-stained agarose gels (data not shown). To determine whether Sp1
could directly modulate apoptosis, Sp1 cDNA was
transiently overexpressed in WKY3M-22 cells using a cytomegaloviral
promoter-driven expression vector (CMV-Sp1). Sp1 increased apoptosis in
this cell type (Fig. 1B). Conversely, apoptosis was
inhibited by 50% in WKY12-22 cells following overexpression of a
dominant-negative form of Sp1 (DNA binding domain) using pEBGNLS-Sp1
(33) (Fig. 1C), demonstrating profound inhibition effected
by a single transcription factor. These data provide the first
demonstration of the capacity of Sp1 to modulate apoptosis in any cell
type.
Inspection of the FasL promoter sequence revealed the existence of a
putative recognition element for Sp1 (5'-GGGCGG-3') located at
nucleotides Sp1 Binds and Activates the FasL Promoter--
To determine
whether FasL is under the transcriptional control of Sp1, transient
transfection analysis was performed with the construct FasL·hsLuc, a
firefly luciferase-based reporter vector driven by 1.2 kilobases of the
FasL promoter (16). Cotransfection of FasL·hsLuc with an Sp1
expression vector induced FasL promoter-dependent expression
(Fig. 2A). To localize the Sp1
response element in the FasL promoter, we generated a series of
reporter constructs derived from parent FasL·hsLuc bearing 5'
deletions in the FasL promoter. Luciferase activity increased upon
cotransfection of Inducible Apoptosis Involves the Phosphorylation of Sp1 and
Induction of FasL--
We next explored the effect of extracellular
apoptotic stimuli on the capacity of Sp1 to stimulate apoptosis and
transactivate the FasL promoter. CAM, an inhibitor of DNA topoisomerase
I, has been reported to induce apoptosis in several cell types (34) although its effect on SMCs is not known. Nuclear condensation of SMCs
stained by propidium iodide increased dramatically following 24-h
exposure to CAM (Fig. 3A).
This agent also induced internucleosomal fragmentation of DNA and
annexin V staining (data not shown). To determine whether FasL
expression is altered by CAM and define the involvement of Sp1 in this
process, we performed RT-PCR and transient transfection analysis with
FasL promoter constructs. CAM stimulated FasL promoter activity (Fig.
3B) and endogenous FasL gene expression (Fig. 1D)
by 2-4-fold (Fig. 3, B and C). CAM failed to
activate the construct
EMSA using [32P]FasL oligonucleotide and nuclear extracts
of cells exposed to CAM revealed that this agent did alter Sp1
occupancy of the promoter (Fig.
4A, lane 4 versus lane 2). Incubation of these extracts with CIP (22),
which hydrolyzes 5'-phosphate groups, prior to EMSA decreased the
intensity of both Sp1 binding complexes (Fig. 4A, lane
5 versus lane 3) but was most profound in extracts of
cells exposed to CAM. Densitometric assessment of the intensities of
these complexes (Fig. 4A, lower left and lower center) revealed that 12% of promoter-bound Sp1 is
basally phosphorylated and that Sp1 phosphorylation increases to 31%
upon exposure to CAM (Fig. 4A, lower right). This
indirect determination of Sp1 phosphorylation was supported by Western
immunoblot analysis with antibodies to Sp1. We observed the appearance
of a hyperphosphorylated species following exposure to CAM (Fig.
4B). This effect was abolished by prior incubation of the
extracts with CIP (Fig. 4B). These findings thus show that
Sp1 is phosphorylated during CAM-inducible apoptosis. Sp1
phosphorylation regulates the inducible expression of a number of other
genes, including vascular permeability factor/vascular endothelial
growth factor (31), CAM-inducible FasL Promoter Activity and Apoptosis Are Protein
Kinase-
Fas receptor, unlike FasL (Fig. 1C), is expressed in both
WKY12-22 and WKY3M-22 cells (Fig.
6A). Because CAM induces FasL expression (Figs. 3, B-D, and 5A), we
hypothesized that the induction of apoptosis by this agent involves the
secretion and autocrine/paracrine engagement of FasL with Fas at the
cell surface. To address this possibility, prior to the addition of
CAM, we incubated the cells with Fas·Fc chimera, in which the
extracellular domain of Fas is fused to the Fc portion of human IgG.
Fas·Fc blocked SMC apoptosis induced by CAM (Fig. 6B). In
contrast, an identical amount of the Fc fragment alone had no effect
(Fig. 6B). These findings thus demonstrate that
autocrine/paracrine extracellular Fas/FasL engagement is involved in
SMC apoptosis. Sp1 is phosphorylated and activates FasL in SMCs upon
exposure to extracellular apoptotic stimuli.
In this paper, we have defined a novel role for the ubiquitously
expressed transcription factor Sp1 in apoptotic signal transduction. Subtypes of SMC expressing abundant levels of Sp1 produce FasL and
undergo greater spontaneous apoptosis. EMSA and transient transfection
analysis revealed that the FasL promoter is activated by Sp1 via a
distinct element whose integrity is crucial for inducible expression.
Inducible FasL transcription is inhibited by a mutant form of Sp1,
which also blocks apoptosis. Inducible SMC apoptosis is preceded by Sp1
phosphorylation, increased FasL transcription, and the
autocrine/paracrine engagement of FasL with Fas. Both inducible FasL
transcription and apoptosis are blocked by dominant-negative protein
kinase C-
The present study is the first report of transcription factor
phosphorylation as a prerequisite biochemical process in inducible apoptotic cell death. We used CAM as a model effector of cell death;
however, given the general cellular expression of Sp1, our observations
are unlikely to be confined to this agent alone nor are they likely to
be cell type-specific. Okadaic acid, a selective inhibitor of
serine-threonine phosphatase PP2A, stimulates apoptosis in a wide
variety of cell types including murine fibroblasts (41), rat kidney
epithelial cells (42) amongst them. Okadaic acid, like CAM,
stimulates Sp1 phosphorylation and apoptosis in SMCs (data not shown).
Tat, the transcriptional activator of human immunodeficiency virus type
1 (HIV-1), stimulates Sp1 phosphorylation (43), activates FasL
expression (44), and can induce apoptosis (44, 45). Sp1 phosphorylation
may be an important theme in apoptotic signaling. However, this process
alone may not account for FasL transactivation because Sp1
physically interacts and functionally cooperates with a large number of
other transcription factors. These include NF-
A common pathophysiologic setting in which Sp1 phosphorylation may be
relevant is atherosclerosis. Sudden death in patients with unstable
angina and myocardial infarction is associated with atherosclerotic
plaque rupture (49, 50). This could arise from SMC apoptosis, because
SMCs are the only cells in the plaque capable of producing collagen
fibers types I and III, which maintain tensile strength (8-10, 12,
51). Loss of SMCs as a consequence of apoptosis could weaken the cap
and result in plaque rupture (12). Consistent with this, SMCs located
in vulnerable regions of the plaque, such as the fibrous cap and
shoulders (52, 53) undergo apoptosis (56, 54) and express Fas (5, 7).
Indeed, SMCs isolated from atherosclerotic plaques undergo greater
spontaneous apoptosis than cells derived from the normal artery wall
(55). Moreover, SMC depletion in human aortic aneurysms is accompanied by biochemical and morphological changes consistent with SMC
apoptosis (56). Sp1 phosphorylation may thus be an important
biochemical mediator of cell death in a number of vascular disease settings.
, whose wild-type counterpart
phosphorylates Sp1. Thus, Sp1 phosphorylation is a proapoptotic
transcriptional event in vascular SMC and, given the wide distribution
of this housekeeping transcription factor, may be a common regulatory
theme in apoptotic signal transduction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B (16), AP-1 (16), NFAT
(17), ATF2 (18), Egr-2 (17), and Egr-3 (17). The promoter contains a
single transcription initiator site, as well as positive and negative
regulatory regions within a 2.3-kilobase portion of the 5'-untranslated
genome (19). Analysis of the FasL promoter has mostly been confined to
T cells. For example, T cell activation following CD4 cross-linking
induces NFAT binding to the FasL enhancer and gene transactivation
(19). Similarly, cytotoxic stress-induced FasL expression involves the activation of NF-
B, AP-1, and c-Jun N-terminal kinase, prior to cell
death (16-20). Activity of the FasL promoter is also regulated by MEK
kinase-1 (18). However, transcription factor phosphorylation has not
yet been directly demonstrated as a prerequisite step in apoptosis.
281/
276 base pairs (GGGCGG) relative
to the transcriptional start site. Sp1 can influence gene expression by
changes in its nuclear concentration and interaction with the promoter,
by providing architectural support, serving as a cofactor, or by
undergoing chemical modification. Sp1 phosphorylation e.g.
mediates inducible tissue factor expression in vascular endothelial
cells exposed to fluid shear stress (23). The significance of Sp1 in
the process of apoptosis in any cell type is presently unknown. This
knowledge would advance our understanding of the transcriptional basis
of extrinsic apoptosis, given the wide distribution of both Sp1 and
FasL.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
271FasL·hsLuc and
296FasL·hsLuc) were amplified from
the parent vector FasL·hsLuc (gift of Dr Shailaja Kasibhatla,
La Jolla Inst. of Cellular Immunology) by PCR and blunt-end cloned into
pGL3. The mutant counterpart of FasL·hsLuc bearing a mutation in the
Sp1 binding site (mSp1FasL·hsLuc) was constructed using the
QuikChange site-directed mutagenesis kit (Stratagene). pEBGNLS-Sp1 was
obtained from Gerald Thiel (Inst. for Genetics, University of Cologne),
CMV-Sp1 was obtained from Robert Tjian (Howard Hughes Medical Inst.,
University of California), and CMV-FLAG·DN-PKC-
was obtained from
Debabrata Mukhopadhyay (Beth Israel-Deaconess Hospital, Boston) and
Alex Toker (Boston Biomedical Research Inst.).
80 °C until use. All buffers contained protease inhibitors.
80 °C.
-actin are as follows: 5'-FasL, 5'-AAACCCTTTCCTGGGGC-3';
3'-FasL, 5'-GTGTCTTCCCATTCCAG-3'; 5'-Fas,
5'-CTGTGGATCATGGCTGTCCTGCCT-3'; 3'-Fas,
5'-CTCCAGACTTTGTCCTTCATTTTTC-3'; 5'-
-actin,
5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; 3'-
-actin, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'; 5'-glyceraldehyde-3-phosphate dehydrogenase, 5'-ACCACAGTCCATGCCATCAC-3';
3'-glyceraldehyde-3-phosphate dehydrogenase,
5'-TCCACCACCCTGTTGCTGTA-3'.
-actin primers using a Perkin Elmer thermocycler. Amplication was
performed by denaturing the sample at 94 °C for 2 min, then cycled
at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for
30 s, and an extension at 72 °C for 4 min. The number of PCR cycles for Fas/
actin was 25. Twenty microliters of the reaction was
visualized on 1.5% agarose gels with ethidium bromide staining.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Differences in spontaneous apoptosis and FasL
expression in WKY12-22 and WKY3M-22 cells. A,
spontaneous levels of apoptosis as determined by internucleosomal DNA
fragmentation (left) and annexin V/FACS analysis
(right) in WKY12-22 and WKY3M-22 cells. The y
axis in the left and right panels represents
total internucleosomal DNA fragmentation (as a proportion of the cell
population) and annexin V staining (as a percentage of the total number
of cells in the population), respectively. B, overexpression
of Sp1 in WKY3M22 cells stimulates apoptosis. SMCs were transfected
with 0, 1, 3, and 5 µg of CMV-Sp1 using FuGENE6 prior to
quantification of apoptosis after 24 h by ELISA. Where required,
the total amount of DNA transfected was supplemented to 5 µg with
pcDNA3. C, dominant-negative Sp1 blocks apoptosis in
WKY12-22 cells. SMCs were transfected with 40 µg of pEBGNLS or
pEBGNLS-Sp1 using FuGENE6 prior to quantification of apoptosis after
24 h by ELISA. Results in B and C
(ordinate axes) are expressed as total internucleosomal DNA
fragmentation as a proportion of the total number of cells in the
treatment population. D, WKY12-22 cells, but not WKY3M-22
cells, express FasL mRNA, and this is stimulated by CAM. Total RNA
prepared from WKY12-22 cells incubated with CAM (1 µg/ml) for 24 h prior to reverse-transcription and PCR with the indicated primers.
PCR cycle number is indicated in the figure. The densitometric
assessment of the amplicons is represented histodiagramatically.
E, comparative FasL mRNA expression in WKY12-22 cells
and WKY3M-22 cells. Northern blot analysis was performed with total RNA
of WKY3M-22 cells or WKY12-22 cells (with or without 24-h incubation
with 1 µg/ml CAM. The data are representative of two independent
determinations. Error bars represent S.E.
281/
276. Because Sp1 is preferentially expressed in
WKY12-22 cells (26), we hypothesized that FasL, if it is Sp1-dependent, would be more abundantly expressed in
WKY12
22 cells than WKY3M-22 cells. We assessed FasL mRNA
expression in WKY12
22 and WKY3M-22 cells by semiquantitative RT-PCR.
FasL was readily expressed in WKY12-22 cells but was weakly, if at all, expressed in WKY3M-22 cells (Fig. 1D). Northern blot
analysis confirmed preferential FasL mRNA expression in WKY12-22
cells (Fig. 1E). These findings suggested that Sp1 may
regulate cell death through its activation of the FasL promoter.
296FasL·hsLuc and CMV-Sp1 (Fig. 2B).
In contrast,
271FasL·hsLuc failed to respond to Sp1 overexpression
(Fig. 2B). The 5'-FasL promoter end points in these
constructs (
296 and
271) occur on either side of the putative Sp1
binding site (
281/
276). EMSA using a 32P-labeled
double-stranded oligonucleotide spanning this region of the promoter
([32P]FasL oligonucleotide,
296/
265) and nuclear
extracts prepared from WKY12-22 cells revealed the formation of a
number of nucleoprotein complexes (Fig. 2C). The most
intense band was supershifted with polyclonal antibodies directed to
Sp1 (Fig. 2C). Identical amounts of Smad1 polyclonal
antibodies used as a negative control had no influence on nucleoprotein
complex formation (Fig. 2C). To demonstrate sequence
specificity of complex formation, we prepared an oligonucleotide
([32P]mFasL oligonucleotide,
296/
265) bearing a
mutation that disrupts the Sp1 binding site (to 5'-TTTCTT-3'). This
mutation no longer supported the interaction of Sp1 with this region of
the FasL promoter (Fig. 2C). When introduced into
full-length FasL·hsLuc, producing construct mSp1FasL·hsLuc,
luciferase expression inducible by Sp1 was completely abolished (Fig.
2D). These findings demonstrate that Sp1 positively
regulates FasL transcription. Activation by exogenous Sp1 of the FasL
promoter was greater in WKY3M-22 cells than WKY12-22 cells
(Fig. 2, E versus A), consistent with higher endogenous Sp1 expression in the latter cell type (26).
View larger version (29K):
[in a new window]
Fig. 2.
Sp1 activates the FasL promoter in SMCs.
Transient cotransfection analysis in WKY12-22 cells overexpressing Sp1
with FasL-hsLuc (A) and derivatives of FasL·hsLuc bearing
5' deletions in the FasL promoter (B). 6 µg of pcDNA3
or CMV-Sp1 was used in B; 15 µg of FasL promoter-reporter
construct was used throughout. Firefly luciferase activity was
normalized to Renilla activity and the results were plotted
as -fold increase relative to 296FasL·hsLuc or
271FasL·hsLuc,
respectively. C, EMSA using [32P]FasL Oligo,
[32P]mFasL Oligo, and nuclear extracts of WKY12-22 cells.
EMSA was performed as described under "Experimental Procedures,"
and nucleoprotein complexes were visualized by autoradiography.
Arrows indicate nucleoprotein complexes, S
denotes a supershift. Sequence of [32P]mFasL Oligo
(
296/
265) is 5'-ATCAGAAAATTGTGGGCGGAAACTTCCAGG-3', and
[32P]mFasL Oligo is
5'-ATCAGAAAATTGTTTTCTTAAACTTCCAGG-3';
the mutation is underlined). D, mutation
of the Sp1 site in FasL·hsLuc abrogates activation of the FasL
promoter. E, Sp1 activation of FasL promoter in WKY3M-22
cells. 3 or 6 µg of pcDNA3 or CMV-Sp1 were used in cotransfection
experiments with 15 µg of FasL promoter-reporter construct
throughout. Firefly luciferase activity was normalized to
Renilla activity, and the results were plotted as -fold
increase relative to the pcDNA3 control in the FasL·hsLuc and
mSp1FasL·hsLuc contransfectant groups, respectively. Error
bars represent S.E. The data are representative of two independent
determinations.
271FasL·hsLuc (Fig. 3B), which
lacks the Sp1 site and was unable to mediate Sp1-inducible FasL
promoter-dependent expression (Fig. 2B).
Overexpression of dominant-negative Sp1 (20) together with FasL·hsLuc
blocked CAM-inducible FasL transcription (Fig. 3C), whereas
the empty expression vector had no effect (Fig. 3C). These
data demonstrate that Sp1 is required for FasL promoter activation by
extracellular stimuli.
View larger version (48K):
[in a new window]
Fig. 3.
CAM stimulates apoptosis in vascular SMCs and
induces FasL expression in an Sp1-dependent manner.
A, CAM increases propidium iodide nuclear staining in
WKY12-22 cells. The SMCs were exposed to CAM (1 µg/ml) for 24 h
prior to fixation, incubation with propidium iodide, and confocal
microscopy. Effect of CAM (1 µg/ml) on luciferase expression driven
by FasL·hsLuc or a construct bearing a deletion of the Sp1 binding
element ( 271FasL·hsLuc, B), or FasL·hsLuc
cotransfected with 5 µg of dominant-negative Sp1 (pEBGNLS-Sp1) or the
backbone alone (pEBGNLS, C). 15 µg of FasL
promoter-reporter construct was used throughout. Firefly luciferase
activity was normalized to Renilla activity, and the results
were plotted as -fold increase relative to FasL·hsLuc or
271FasL·hsLuc, respectively, in B, or the pEBGNLS or
pEBGNLS-Sp1 groups in C. Error bars represent
S.E. The data are representative of two independent
determinations.
2-integrin (35), and tissue factor
(23).
View larger version (27K):
[in a new window]
Fig. 4.
Phosphorylation of endogenous Sp1 following
exposure to CAM. A, EMSA using
[32P]mFasL Oligo and nuclear extracts of WKY12-22 cells
incubated with CAM (1 µg/ml). Where appropriate, the extracts were
treated with CIP prior to EMSA. The amount of CIP (5 milliunit) used in
this assay is based on CIP titration experiments that previously
defined the concentration of CIP unable to dephosphorylate the
32P-labeled probe. Sp1 nucleoprotein complex intensity
(with or without CAM exposure for CIP treatment of extracts) was
semi-quantitated by densitometry. B, Western blot analysis
using nuclear extracts of WKY12-22 cells exposed to CAM (1 µg/ml),
with and without CIP treatment (5 units). Sp1-P indicates
hyperphosphorylated Sp1. The Coomassie Blue-stained gel is shown. The
data are representative of two independent determinations.
-dependent Processes--
A kinase found to
mediate Sp1 phosphorylation is protein kinase-
(PKC-
) (31), a
diacylglycerol- and Ca2+-independent atypical member of the
PKC family (36). PKC-
is ubiquitously expressed and interacts
directly with Sp1 (31). To investigate the role of PKC-
in the
regulation of FasL expression, we cotransfected WKY12-22 cells with an
expression vector (CMV-FLAG·DN-PKC-
) generating a kinase-inactive
dominant-negative mutant of PKC-
bearing a
Lys275
Trp275 substitution (37-40). The FasL
promoter was activated by CAM in cells harboring the empty expression
vector (Fig. 5A), whereas overexpression of mutant PKC-
attenuated CAM-inducible FasL
promoter-dependent reporter expression (Fig.
5A). Dominant-negative PKC-
also blocked internucleosomal
fragmentation stimulated by CAM (Fig. 5B). Overexpression of
dominant negative PKC-
in cells not exposed to CAM did not significantly modulate the level of apoptosis compared with cells transfected with the backbone control (data not shown). This suggests that the capacity of dominant-negative PKC-
to suppress apoptosis detectable in our system is conditional upon the cells being induced to
undergo further cell death by exposure to apoptotic stimuli. This is
likely a direct consequence of the low level of spontaneous phosphorylation of Sp1 (Fig. 4A) making attenuation by
dominant-negative PKC-
difficult to measure in a cotransfection
setting. These findings, nonetheless, indicate that PKC-
regulates
inducible FasL transcription and apoptosis.
View larger version (14K):
[in a new window]
Fig. 5.
CAM induction of the FasL promoter and
apoptosis are PKC- -dependent
processes. A, WKY12-22 cells were transfected with
FasL·hsLuc and 3 µg of either CMV-FLAG or CMV-FLAG·DN-PKC-
prior to determination of luciferase activity after 24 h. Firefly
luciferase activity was normalized to Renilla activity, and
the results were plotted as -fold increase relative to the CMV-FLAG or
CMV-FLAG·DN-PKC-
groups, respectively. B, WKY12-22
cells were transfected with 3 µg of either CMV-FLAG or
CMV-FLAG·DN-PKC-
. 15 µg of FasL promoter-reporter construct was
used throughout. After 24 h, the cells were incubated with CAM (1 µg/ml) for a further 24 h, and apoptosis was assessed by ELISA.
Results (ordinate axis) are expressed as total
internucleosomal DNA fragmentation as a proportion of the total number
of cells in the treatment population. Error bars represent
S.E. The data are representative of two independent
determinations.
View larger version (18K):
[in a new window]
Fig. 6.
Autocrine/paracrine FasL-Fas engagement
mediates CAM-inducible apoptosis in SMCs. A,
determination of spontaneous Fas expression in WKY12-22 and WKY3M-22
cells by RT-PCR. B, CAM-inducible apoptosis is blocked by
Fas-Fc. WKY12-22 cells were exposed to 50 µg/ml Fas·Fc or Fc for
1 h and then incubated with CAM (0.1 µg/ml) for 24 h with
assessment of apoptosis by ELISA. Results (ordinate axis)
are expressed as total internucleosomal DNA fragmentation as a
proportion of the total number of cells in the treatment population.
Error bars represent S.E. The data are representative of two
independent determinations.
. These data demonstrate that apoptotic signaling in SMCs
involves Sp1 phosphorylation.
B p65/RelA (46, 47)
and AP1 (48), which each induce the FasL promoter in other cell types
(16, 19).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Miss Onza Chan for
excellent technical assistance and Drs. Shailaja Kasibhatla (La Jolla
Institute of Cellular Immunology) for the construct FasL·hsLuc,
Gerald Thiel (Institute for Genetics, University of Cologne) for
pEBGNLS-Sp1, Robert Tjian (Howard Hughes Medical Institute, University
of California) for CMV-Sp1, Debabrata Mukhopadhyay (Beth
Israel-Deaconess Hospital, Boston) and Alex Toker (Boston Biomedical
Research Institute) for CMV-FLAG·DN-PKC-.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Australian Research Council (to L. M. K.), National Health and Medical Research Council of Australia (NHMRC) (to L. M. K.), and an NSW Department of Health Infrastructure grant to the Centre for Thrombosis and Vascular 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.
¶ Research Fellow of the NHMRC.
To whom correspondence should be addressed. Tel.: 61-2-9385 2537; Fax: 61-2-9385 1389; E-mail: l.Khachigian@unsw.edu.au.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009251200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FasL, Fas ligand;
CIP, calf intestinal alkaline phosphatase;
DN-PKC-, dominant-negative protein kinase C-
;
SMC, smooth muscle cells;
CAM, camptothecin;
PCR, polymerase chain reaction;
RT-PCR, reverse
transcription-PCR;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked
immunosorbent assay;
FACS, fluorescence-activated cell sorter;
EMSA, electrophoretic mobility shift assay;
CMV, cytomegalovirus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Ashkenazi, A.,
and Dixit, V. M.
(1998)
Science
281,
1305-1308 |
2. | Ashkenazi, A., and Dixit, V. M. (1999) Curr. Opin. Cell Biol. 11, 255-260[CrossRef][Medline] [Order article via Infotrieve] |
3. | Zhang, J., Cado, D., Chen, A., Kabra, N. H., and Winoto, A. (1998) Nature 392, 296-300[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Hirata, H.,
Takahashi, A.,
Kobayashi, S.,
Yonehara, S.,
Sawai, H.,
Okazaki, T.,
Yamamoto, K.,
and Sasada, M.
(1998)
J. Exp. Med.
187,
587-600 |
5. | Geng, Y. J., Liao, H.-S., and Macgovern, J. (1998) Circulation 98 (suppl. I), I-48 |
6. |
Geng, Y. J.,
Henderson, L. E.,
Levesque, E. B.,
Muszynski, M.,
and Libby, P.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
2200-2208 |
7. | Cai, W., Devaux, B., Schaper, W., and Schaper, J. (1997) Atherosclerosis 131, 177-186[CrossRef][Medline] [Order article via Infotrieve] |
8. | Kockx, M. M., and Herman, A. G. (1998) Eur. Heart J. 19, G23-G28[Medline] [Order article via Infotrieve] |
9. |
Kockx, M. M.,
de Meyer, G. R. Y.,
Muhring, J.,
Jacob, W.,
Bult, H.,
and Herman, A. G.
(1998)
Circulation
97,
2307-2315 |
10. |
Kockx, M. M.
(1998)
Arterioscler. Thromb. Vasc. Biol.
18,
1519-1522 |
11. |
Flynn, P. D.,
Byrne, C. D.,
Baglin, T. P.,
Weissberg, P. L.,
and Bennett, M. R.
(1997)
Blood
89,
4378-4384 |
12. | Kockx, M. M., and Herman, A. G. (2000) Cardiovasc. Res. 45, 736-746[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Sata, M.,
Perlman, H.,
Muruve, D. A.,
Silver, M.,
Ikebe, M.,
Libermann, T. A.,
Oettgen, P.,
and Walsh, K.
(1998)
Proc. Natl. Acad. Sci. U S A
95,
1213-1217 |
14. |
Luo, Z.,
Sata, M.,
Nguyen, T.,
Kaplan, J. M.,
Akita, G. Y.,
and Walsh, K.
(1999)
Circulation
99,
1776-1779 |
15. |
Schnieder, D. B.,
Vassalli, G.,
Wen, S.,
Driscoll, R. M.,
Sassani, A. B.,
DeYong, M. B.,
Linnemann, R.,
Zvirmani, R.,
and Dichek, D. A.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
298-308 |
16. | Kasibhatla, S., Brunner, T., Genestier, L., Echeverri, F., Mahboubi, A., and Green, D. R. (1998) Mol. Cell 1, 543-551[Medline] [Order article via Infotrieve] |
17. | Xiao, S., Matsui, K., Fine, A., Zhu, B., Marshak-Rothstein, A., Widom, R. L., and Ju, S.-T. (1999) Eur. J. Immunol. 29, 3456-3465[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Faris, M.,
Latinis, K. M.,
Kempiak, S. J.,
Koretzky, G. A.,
and Nel, A.
(1998)
Mol. Cell. Biol.
18,
5414-5424 |
19. |
Holtz-Heppelmann, C. J.,
Algeciras, A.,
Badley, A. D.,
and Paya, C. V.
(1998)
J. Biol. Chem.
273,
4416-4423 |
20. |
Faris, M.,
Kokot, N.,
Latinis, K.,
Kasibhatla, S.,
Green, D. R.,
Koretzky, G. A.,
and Nel, A.
(1998)
J. Immunol.
160,
134-144 |
21. | Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 5, 155-165[CrossRef] |
22. | Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987) Cell 51, 1079-1090[Medline] [Order article via Infotrieve] |
23. |
Lin, M.-C.,
Almus-Jacobs, F.,
Chen, H.-H.,
Parry, G. C. N.,
Mackmann, N.,
Shyy, J. Y.,
and Chen, S.
(1997)
J. Clin. Invest.
99,
737-744 |
24. | Majesky, M. W., Giachelli, C. M., Reidy, M. A., and Schwartz, S. M. (1992) Circ. Res. 71, 759-768[Abstract] |
25. | Lemire, J. M., Covin, C. W., White, S., Giachelli, C. M., and Schwartz, S. M. (1994) Am. J. Pathol. 144, 1068-1081[Abstract] |
26. |
Rafty, L. A.,
and Khachigian, L. M.
(1998)
J. Biol. Chem.
273,
5758-5764 |
27. |
Ho, F. M.,
Liu, S. H.,
Liau, C. S.,
Huang, P. J.,
and Lin-Shiau, S.
(2000)
Circulation
101,
2618-2624 |
28. | Jones, M. M., Xu, C., and Ladd, P. A. (1997) Toxicolology 116, 169-175[CrossRef][Medline] [Order article via Infotrieve] |
29. | Sandoval, M., Zhang, X.-J., Liu, X., Mannick, E. E., Clarck, D. A., and Miller, M. J. S. (1997) Free Red. Biol. Med. 22, 489-495[CrossRef] |
30. |
Leggett, R. W.,
Armstrong, S. A.,
Barry, D.,
and Mueller, C. R.
(1995)
J. Biol. Chem.
270,
25879-25884 |
31. |
Pal, S.,
Claffey, K. P.,
Cohen, H. T.,
and Mukhopadhyay, D.
(1998)
J. Biol. Chem.
273,
26277-26280 |
32. |
Khachigian, L. M.,
Williams, A. J.,
and Collins, T.
(1995)
J. Biol. Chem.
270,
27679-27686 |
33. | Petersohn, D., and Thiel, G. (1996) Eur. J. Biochem. 239, 827-834[Abstract] |
34. | Pantazis, P. (1995) Leukemia Res. 19, 775-788[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Zutter, M. M.,
Ryan, E. E.,
and Painter, A. D.
(1997)
Blood
90,
678-689 |
36. |
Marshall, M. S.
(1995)
FASEB J.
9,
1311-1318 |
37. | Huang, C., Ma, W., and Dong, Z. (1997) Oncogene 14, 1945-1954[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Bandyopadhyay, G.,
Standaert, M. L.,
Zhao, L., Yu, B.,
Avignon, A.,
Galloway, L.,
Karnam, P.,
Moscat, J.,
and Farese, R. V.
(1997)
J. Biol. Chem.
272,
2551-2558 |
39. | van Dijk, M., Muriana, F. J., van der Hoeven, P. C., de Widt, J., Schaap, D., Moolenaar, W. H., and van Blitterswijk, W. J. (1997) Biochem. J. 323, 693-699[Medline] [Order article via Infotrieve] |
40. |
Standaert, M. L.,
Galloway, L.,
Karnam, P.,
Bandyopadhyay, G.,
Moscat, J.,
and Farese, R. V.
(1997)
J. Biol. Chem.
272,
30075-30082 |
41. | D'Ambrosio, C., Valentinis, B., Prisco, M., Reiss, M., Rubini, M., and Baserga, R. (1997) Cancer Res. 57, 3264-3271[Abstract] |
42. | Davis, M. A., Smith, M. W., Chang, S. H., and Trump, B. F. (1994) Toxicol. Pathol. 22, 595-605[Medline] [Order article via Infotrieve] |
43. |
Chun, R. F.,
Semmes, O. J.,
Neuveut, C.,
and Jeang, K. T.
(1998)
J. Virol.
72,
2615-2629 |
44. | Westendorp, M. O., Frank, R., Ochsenbauer, C., Stricker, K., Dhein, J., Walczak, H., Debatin, K. M., and Krammer, P. H. (1995) Nature 375, 497-500[CrossRef][Medline] [Order article via Infotrieve] |
45. | Li, C. J., Friedman, D. J., Wang, C., Metelev, V., and Pardee, A. B. (1995) Science 268, 429-431[Medline] [Order article via Infotrieve] |
46. | Perkins, N. D., Edwards, N. L., Duckett, C. S., and Agranoff, A. B. (1993) EMBO J. 12, 3551-3558[Abstract] |
47. | Perkins, N. D., Agranoff, A. B., Pascal, E., and Nabel, G. J. (1994) Mol. Cell. Biol. 14, 6570-6583[Abstract] |
48. |
Li, Y.,
Mak, G.,
and Franza, B. R.
(1994)
J. Biol. Chem.
269,
30616-30619 |
49. | Davies, M. J., Richardson, P. D., Woolf, N., Katz, D. R., and Mann, J. (1993) Br. Heart. J. 69, 377-381[Abstract] |
50. | van der Wal, A. C., Becker, A. E., van der Loos, C. M., and Das, P. K. (1994) Circulation 89, 36-44[Abstract] |
51. | Newby, A. C. (1997) Coron. Artery Dis. 8, 213-224[Medline] [Order article via Infotrieve] |
52. | Richardson, P. D., Davies, M. J., and Born, G. V. R. (1989) Lancet 2, 941-944[Medline] [Order article via Infotrieve] |
53. | Cheng, G. C., Loree, H. M., Kamm, R. D., Fishbein, M. C., and Lee, R. T. (1993) Circulation 87, 1179-1187[Abstract] |
54. | Bjorkerud, S., and Bjorkerud, B. (1996) Am. J. Pathol. 149, 367-380[Abstract] |
55. |
Bennett, M. R.,
Littlewood, T. D.,
Schwartz, S. M.,
and Weissberg, P. L.
(1997)
Circ. Res.
81,
591-599 |
56. | Thompson, R. W., Liao, S., and Curci, J. A. (1997) Coron. Artery Dis. 8, 623-631[Medline] [Order article via Infotrieve] |