Ms. #H0917-8 (Revision #1)
Vascular
smooth
muscle
cell
growth
arrest
upon
1
blockade
of
thrombospondin-1 requires p21Cip1/WAF1.
Donghui Chen ‡, Kun Guo ‡, Jihong Yang ‡, William A. Frazier *, Jeffrey M. Isner
‡
and Vicente
Andrés ‡ †.
‡
Department of Medicine (Cardiology), St. Elizabeth’s Medical Center, Tufts University School
of Medicine, Boston, MA, 02135; † Instituto de Biomedicina de Valencia, Consejo Superior de
Investigaciones Científicas, 46010-Valencia, Spain; and *Department of Biochemistry and
Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110
Running title: Anti-TSP1 antibody inhibits cell growth in a p21-dependent manner
Corresponding author:
Vicente Andrés, Ph.D.
Division of Cardiovascular Research
St. Elizabeth’s Medical Center of Boston
736 Cambridge Street
Boston, MA 02135
Phone: 617-562-7509
Fax: 617-562-7506
E-mail: vandres@ibv.csic.es
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Abstract
Abnormal proliferation of vascular smooth muscle cells (VSMCs) is thought to play an
important role in the pathogenesis of atherosclerosis and restenosis. Previous studies have
implicated the extracellular matrix protein thrombospondin-1 (TSP1) in mitogen-dependent
proliferation of VSMCs. In this study, we investigated the molecular mechanisms involved in
TSP1-mediated regulation of VSMC growth. Neutralizing A4.1 anti-TSP1 antibody inhibited the
activity of the G1/S cyclin-dependent kinase 2 (cdk2) and blocked the induction of S-phase entry
which normally occurs in serum-stimulated VSMCs. This growth inhibitory effect was associated
with a marked induction of p21Cip1/WAF1 (p21) expression in A4.1-treated VSMCs. Moreover,
addition of A4.1 antibody to VSMCs markedly increased the level of p21 bound to cdk2. Thus,
growth arrest upon antibody blockade of TSP1 may be mediated by the cdk inhibitory protein
p21. Consistent with this notion, anti-TSP1 antibody inhibited [3H]-thymidine incorporation in
wild-type, but not in p21-deficient mouse embryonic fibroblasts (MEFs). Taken together, these
data suggest that p21 plays an important role in TSP1-mediated control of cellular proliferation.
KEY WORDS: vascular smooth muscle cells, cell cycle control, p21, thrombospondin,
extracellular matrix.
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Introduction
The extracellular matrix (ECM) plays a critical role in highly specialized cellular
functions, including differentiation, migration, and proliferation (5, 21, 45, 46). The composition
and structure of the ECM differ from tissue to tissue and can undergo continuous changes within
the same tissue, thereby having both temporal and spatial effects on cells that come to contact
with it. These changes result in part from the regulation of the synthesis and secretion of the
glycoproteins that are incorporated into the ECM.
Unlike terminally differentiated myocytes, mature smooth muscle cells can reenter the
cell cycle in response to physiopathological stimuli (35). Dedifferentiation and proliferation of
vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of vascular occlusive
disease, including atherosclerosis, restenosis after angioplasty and bypass graft occlusion.
Inhibition of VSMC proliferation has been shown to attenuate restenosis following balloon
angioplasty in several animal models (12, 50). VSMC proliferation induced by growth factors in
vitro and balloon injury in vivo is associated with changes in the expression of ECM proteins and
their corresponding cellular receptors, which may play an important role as physiological
regulators of cell cycle progression during atherosclerosis and restenosis (2). One of the ECM
components for which dramatic regulatory changes have been observed is thrombospondin-1
(TSP1), a member of a family of related glycoproteins (TSP1 through TSP5) (3, 4, 10, 24, 25, 34,
41). TSP1 is secreted by numerous cell types, including platelets, endothelial cells, macrophages,
fibroblasts and VSMCs (19, 20, 25, 31, 33, 43). TSP1 expression is rapidly upregulated upon
serum or growth factor stimulation of cultured VSMCs (11, 28, 30), and TSP1 protein and
mRNA are elevated with both intimal hyperplasia and hypercholesterolemia in vivo (26, 42-44,
56). While TSP1 appears to be important for the proliferation of VSMCs (27, 29) and fibroblasts
(38), it inhibits endothelial cell growth in vitro (54) and angiogenesis in vivo (13, 18). However,
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the molecular mechanisms by which TSP1 exerts these cell-type specific functions are not well
understood.
Cell cycle progression is facilitated by the sequential activation of a family of cyclindependent kinases (cdks), which requires their association with specific subunits called cyclins
(15, 32, 49). Cdk2 activity is negatively regulated by members of the growth suppressor family of
cdk inhibitors (CKIs), including p21Cip1/WAF1 (p21) and p27Kip1 (p27) (14, 16, 37). In the present
study we investigated the molecular pathways through which TSP1 regulates VSMC
proliferation in vitro by using neutralizing anti-TSP1 monoclonal antibody A4.1. Our results
demonstrate that A4.1-mediated growth arrest in serum-stimulated VSMCs is associated with the
inhibition of cdk2-dependent kinase activity. Expression of p21 and its association with cdk2
complexes was induced upon addition of A4.1 antibody to serum-stimulated VSMCs. Moreover,
A4.1 antibody blocked DNA synthesis in wild-type mouse embryonic fibroblasts (MEFs), but not
in cells derived from p21-deficient mice. Taken together, these data demonstrate that antibody
blockade of TSP1 inhibits cell cycle progression in a p21-dependent manner, and suggest the
involvement of p21 in TSP1-mediated regulation of cellular proliferation.
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Methods
Cell culture. Cells were incubated at 37 0C in a humidified 5% CO295% O2 atmosphere in
medium supplemented with 2 mM L-glutamine, 200 U/ml penicillin, 0.25 mg/ml streptomycin,
and serum as indicated. Primary rat aortic VSMCs were isolated essentially as described (39) and
maintained in DMEM supplemented with 10% FBS (growth medium). MEFs derived from wildtype and p21-deficient mice (8) were maintained in DMEM containing 10% FBS. To enrich the
population of cells in Go/G1, cultures were serum-starved for 3 days in DMEM supplemented
with 0.2% FBS. The C2C12 murine skeletal myoblast cell line was obtained from American
Type Culture Collection. Terminal differentiation of C2C12 myoblasts maintained in 20%
FBS/DMEM was induced by switching cultures to 2% heat-inactivated horse serum/DMEM
(differentiation medium). Under these conditions, C2C12 cells permanently exit the cell cycle
and then express differentiation markers (1).
Anti-TSP1 antibody. The mouse monoclonal anti-TSP1 antibody A4.1, which recognizes the
trypsin-resistant 70 Kd core of TSP1 (40), was used in this study. Specificity of this antibody has
been previously established by Western blotting against samples of whole platelets, serum, and
purified proteins (40). Mouse non-specific IgM antibodies MOPC-104E (M2521) was used as a
control (Sigma Chemical).
[3H]-thymidine uptake analysis. Rat VSMCs and MEFs were plated in 24-well tissue culture
plates in DMEM supplemented with 10% FBS. Cells were rendered quiescent by incubation for 3
days in DMEM containing 0.2% FBS and then cultures were restimulated with growth medium
for 16-24 hours. When indicated, cells were treated with different concentrations of anti-TSP1
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antibody A4.1 (25-100 g IgM per ml of medium). Cells were incubated in growth medium
containing 3 Ci/ml of [3H]-thymidine (6.7 Ci/mmole, Dupont NEN) for the last 4-6 hours. Cells
were washed three times with PBS, and incubated with cold 10% trichloroacetic acid (TCA) for
1 hour. After removing the TCA solution, cells were rinsed three times with water and the
precipitated material was solubilized with 0.25 N NaOH. Tritium content in the sodium
hydroxide solution was determined by adding Scintiverse II (Fisher Scientific) and measured
using a Beckman LS 5000TD scintillation counter. The experiments were performed in triplicate
wells. Parallel cultures of VSMCs and MEFs in 24 well plates were collected by trypsinization
and the cell numbers were determined under microscopy with a hemacytometer.
FACS analysis. Rat VSMCs were plated in 100mm petri dishes in growth medium and allowed
to attach before being transferred to DMEM containing 0.2% FBS. After 72 hours in low serum,
cultures were restimulated by the addition of growth medium. Cells were trypsinized 24 hours
after addition of serum, then washed three times with PBS and fixed in 70% ethanol overnight at
4 0C. DNA was stained with PBS containing 50 µg/ml of each propidium iodide and RNase A
(Boehringer Mannheim). Cell cycle profile was determined at the Core Flow Cytometry Facility
of the Dana Farber Cancer Institute (Boston, MA) using a Beckton Dickinson Vantage flow
cytometer and Lysis II cell cycle analysis software. All experiments were performed in triplicate.
Western blot analysis, immunoprecipitation/western blotting and cdk2-dependent kinase assay.
Subconfluent, starvation-synchronized rat VSMCs (in 100mm plates) were switched to growth
medium with or without the addition of the indicated amounts of either control IgM or A4.1 antiTSP1 antibodies for 16 hours. Cells were washed three times with cold PBS, resuspended in
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500µl of lysis buffer (50mM tris.Cl pH7.4, 150mM NaCl, 1% NP-40, 1mM Na3VO4, 2µg/ml
aproptinin, 2µg/ml leupeptin, 1µM phenylmethylsufonyl floride) and passed through a 26G1/2
needle several times. Insoluble material was cleared by centrifugation at 13,000 rpm for 10
minutes at 4 0C. Protein concentration of lysates was determined using the Bradford reagent
(BioRad Laboratories). 50 µg of protein extract was subjected to electrophoresis on 12% SDSPAGE and transferred to Immobilon-P (Amersham). Membranes were blocked overnight at 4 0C
with buffer A (0.2% Tween-20 in PBS) containing 5% nonfat milk, and then incubated at room
temperature for 3 hours with the indicated primary antibodies diluted in buffer A containing 2%
nonfat milk. The following antibodies were used in this study: anti-p21 (sc-397, 1:200), anti-p27
(sc-528, 1:250), anti-p53 (sc-99, 1:250), anti-cdk2 (sc-163, 1:500), anti-cyclin A (sc-751, 1:200),
and anti-cyclin E (sc-481, 1:250) (Santa Cruz Biotechnology). After several washes with buffer
A, immunocomplexes were detected using an ECL detection kit (Amersham Life Science)
according to the recommendations of the manufacturer. Autoradiographs of Western blots were
scanned and band intensity was determined after background subtraction using a densitometric
program (Sigma Gel, Jandel Scientific).
For immunoprecipitation/Western blot-coupled assays, 200 µg of cell extract was
precleared with 20 µl of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) for 30
0
minutes at 4 C, after which samples were incubated with 2 µg of anti-cdk2 antibodies for 3 h at
4 0C. Immunocomplexes were precipitated with 20 µl of Protein A/G PLUS-Agarose beads at 4
0
C for 1 hour. Pellets were washed three times with lysis buffer and subjected to western blotting
with anti-cdk2 antibodies as described above.
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Cdk2-dependent kinase assays in cell lysates were performed using histone H1
(Boehringer Mannheim) and [32P]ATP (Dupont NEN) substrates as previously described (7).
The reaction mixtures were separated on 12% SDS/PAGE. Gels were stained with Coomassie
blue (Sigma Chemical), dried, and autoradiographed.
Statistics. All results were expressed as mean ± standard error. Statistical significance was
evaluated using ANOVA followed by Scheffe's procedure for more than two means. A value of
p<0.05 was interpreted to indicate statistical significance.
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Results
Neutralizing A4.1 anti-TSP1 antibody blocks serum-inducible DNA synthesis in vascular
smooth muscle cells.
We first investigated the effects of neutralizing A4.1 monoclonal anti-TSP1 antibody on
[3H]-thymidine incorporation upon serum restimulation of starvation-synchronized rat VSMCs.
To this end, cells were incubated for 72 h in 0.2%FBS/DMEM and then stimulated with growth
medium (10%FBS/DMEM) for 24 hours, with or without the addition of A4.1 antibody. As
shown in Figure 1A, serum restimulation of VSMCs treated with control IgM led to ~6-fold
increase in [3H]-thymidine incorporation. However, increasingly higher concentrations of antiTSP1 antibody reduced serum-inducible [3H]-thymidine uptake, with the highest amount of A4.1
antibody tested completely blocking [3H]-thymidine incorporation in serum-restimulated
VSMCs.
We next performed flow cytometry analysis to further characterize the effect of A4.1
antibody on VSMC proliferation. In serum stimulated cultures, approximately 60% and 30% of
the cells were in G1- and S-phase, respectively (Fig. 1B). Whereas treatment of VSMCs with
control IgM did not significantly affect this cell cycle profile, addition of A4.1 antibody
decreased the cell population in S-phase to ~10% and augmented the population in G1 to ~81%.
Thus, consistent with previous studies (29), these data demonstrate that neutralization of TSP1
function in serum-stimulated VSMCs leads to inhibition of DNA synthesis and accumulation of
cells in G1.
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Neutralizing A4.1 anti-TSP1 antibody blocks serum-inducible cdk2-dependent kinase activity
in vascular smooth muscle cells.
Cdk2 function is required for progression through G1- and S-phase (15, 32, 49). When
assayed in vitro using anti-cdk2 antibodies and histone H1 as substrate, cdk2-dependent kinase
activity was downregulated in both starvation-synchronized skeletal muscle cells (SKMCs) and
VSMCs as compared to asynchronously proliferating cells (Fig. 2A, lane Q versus P,
respectively). Consistent with the irreversibility of cell cycle exit in striated myocytes, serumrestimulated SKMCs disclosed impaired induction of cdk2-dependent kinase activity (lane
Q+FBS). In marked contrast, serum-restimulated VSMCs upregulated cdk2-dependent kinase
activity to a level similar to that seen in asynchronously growing cultures, demonstrating that
VSMCs can reversibly regulate cdk2 function in response to mitogens. Since conditions that
promote cell growth arrest were associated with inhibition of cdk2 function, we next sought to
investigate the effect of A4.1 antibody on cdk2-dependent kinase activity in VSMCs. As shown
in Fig. 2B, VSMCs treated with control IgM efficiently upregulated cdk2-dependent kinase
activity upon serum refeeding. In contrast, addition of A4.1 antibody to the medium blocked the
normal serum-dependent induction of cdk2 activity. Thus, inhibition of cdk2 activity may
contribute to the cell cycle inhibitory activity of anti-TSP1 antibodies in VSMCs.
Neutralizing A4.1 anti-TSP1 antibody abrogates serum-inducible cyclin A and cyclin E
expression and induces p21Cip1/WAF1.
Having demonstrated that cell-cycle arrest in A4.1-treated VSMCs is associated with
impaired cdk2 function, we sought to elucidate the molecular mechanisms underlying this
inhibitory effect. As shown by the Western blot analysis of Fig. 3, addition of A4.1 antibody to
serum-restimulated VSMCs had no effect on cdk2 protein levels. Since cdk2 activity during G110
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and S-phase is induced in part through its association with cyclin E and cyclin A, respectively,
the effect of A4.1 antibody on the expression of these regulatory subunits was also studied.
Treatment of serum-restimulated VSMCs with A4.1 antibody blocked serum-inducible
expression of cyclin E and cyclin A (Fig. 3). Thus, inhibition of cdk2 activity by A4.1 is
associated with diminished expression of its cyclin regulatory subunits.
Since cdk2 activity can be negatively regulated through its association with the inhibitory
proteins p21 and p27, we also analyzed the effect of A4.1 antibody on these growth suppressor
molecules. Whereas A4.1 antibody did not affect p27 expression, its addition to serum
restimulated-VSMCs markedly upregulated p21 protein levels (Fig. 3). To test whether the
induction of p21 in A4.1-treated VSMCs resulted in an increased association of p21 with cdk2containing complexes, cell lysates were immunoprecipitated with anti-cdk2 antibodies and then
the immunopellets were subjected to Western blot analysis using anti-p21 antibodies. As shown
in Fig. 3, the abundance of p21 associated with cdk2 was increased by the treatment of cells with
A4.1 antibody. The lack of cdk2-bound p21 in serum restimulated cells that were not treated with
A4.1 antibody (Fig. 3, lanes 2 and 3) may be due to association of p21 with cdk4-cyclin D1
holoenzymes (23, 36). These results suggest that upregulation of p21 and its increased
association with cdk2, together with diminished cyclin A and cyclin E expression, may
contribute to growth arrest in VSMCs treated with neutralizing anti-TSP1 antibody. In these
studies, control mouse IgM had little or no effect on the expression of these cell cycle regulatory
proteins, demonstrating the specificity of the effects elicited by the A4.1 antibody.
p21 is essential for growth suppression upon antibody blockade of TSP1.
The above results suggest that p21 plays an important role on cell cycle arrest in cells
treated with A4.1 antibody. To further investigate the role of p21 on growth arrest induced upon
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3
neutralization of TSP1 function, the effect of A4.1 antibody on [ H]-thymidine incorporation was
tested in MEFs isolated from wild-type and p21-deficient mice. As shown in Fig. 4, addition of
A4.1 antibody inhibited in a dose-dependent manner [3H]-thymidine incorporation in serumstimulated wild-type MEFs. In marked contrast, addition of A4.1 to the culture medium had little
or no effect on [3H]-thymidine uptake in serum-stimulated p21-deficient MEFs. These findings
demonstrate that p21 is essential for the cell cycle inhibitory activity of anti-TSP1 antibody.
Discussion
TSP1 expression is rapidly upregulated upon serum or growth factor stimulation of
quiescent VSMCs (11, 28, 30). Moreover, TSP1 protein and mRNA are detected in VSMCs
within atherosclerotic lesions (26, 43, 44, 56) and during the proliferative response of VSMCs to
vascular injury (42). Consistent with the role of TSP1 as a positive regulator of VSMC
proliferation, addition of neutralizing anti-TSP1 antibodies blocked serum-inducible VSMC
proliferation (29). However, the mechanisms by which TSP1 regulates VSMC growth are not
completely understood. The present study demonstrates that addition of neutralizing A4.1
antibody to cultured VSMCs blocked in a dose-dependent manner the serum-inducible activity of
cdk2, a cell cycle regulator that is required for G1- and S-phase progression. While cdk2
expression was not affected by the addition of A4.1 to VSMC cultures, p21 protein level was
markedly induced in A4.1-treated cells. Importantly, exposure to A4.1 antibody also increased
the interaction of p21 with cdk2 complexes, suggesting that upregulation of p21 contributes to
the repression of cdk2 activity and cell-cycle arrest upon neutralization of TSP1 function. Further
evidence implicating p21 in growth arrest induced by anti-TSP1 antibody was provided using
MEFs derived from either wild-type or p21-null mice. Indeed, A4.1 significantly blocked DNA
synthesis in wild-type, but not in p21-null MEFs. Taken together these data demonstrate that p21
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is essential for A4.1-induced growth arrest, thus implicating p21 as a downstream effector of
TSP1.
Our results show that A4.1 antibody blocks the normal induction of cyclin A and cyclin E
protein expression normally seen in serum-stimulated VSMCs. When taken together with the
requirement of these regulatory subunits for cell-cycle progression (14, 16, 17), these data
suggest that inhibition of cyclin A and cyclin E expression may contribute to growth arrest in
VSMCs exposed to A4.1 antibody. It is noteworthy that serum-dependent induction of cyclin A
promoter activity in VSMCs and fibroblasts requires a functional E2F binding site (47, 52).
Moreover, the CKIs p16, p21 and p27 can repress transcription of E2F target genes, including
cyclin A and cdc2 (7, 9, 48, 52, 59), suggesting that blockade of cyclin A expression in VSMCs
treated with A4.1 antibody may result in part from p21-dependent transcriptional repression.
The effect of CKIs, ECM components and their cellular receptors on VSMC proliferation
has been the subject of recent studies. Expression of p21 and p27 in VSMCs is markedly
upregulated after angioplasty at time points that coincide with the reestablishment of the
quiescent phenotype (7, 53, 58). Moreover, adenovirus-mediated overexpression of p21 (6, 55,
58) and p27 (7) attenuate VSMC hyperplasia after vascular injury in vivo. Regarding the
regulation of CKI expression by specific components of the ECM, Koyama et al. reported that
polymerized type I collagen inhibits mitogen-inducible VSMC proliferation in vitro by
upregulating p27 and p21 levels, while monomer collagen supported cell cycle activity (22).
Interestingly, treatment with neutralizing antibodies to 2 integrins induced p27 and p21
expression and caused cell cycle arrest in VSMCs grown on monomer collagen (22). These
findings suggest that 2 integrins can sense changes in collagen structure that modulate VSMC
proliferation through the regulation of CKI expression. Of note, it has been shown that the TSP
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receptor CD47 (IAP) can associate with 21 integrin and modulate its function in VSMCs (57).
Moreover, TSP-induced VSMC proliferation in vitro is regulated by3 integrins, which are
upregulated during injury-induced VSMC hyperplasia in vivo (51). Thus, regulation of CKI
expression through specific ECM components (i. e., TSP1) appears to be an important regulator
of VSMC growth in vitro.
In summary, the present study demonstrates that VSMC growth arrest upon antibody
blockade of TSP1 is associated with the induction of p21 and repression of cyclin A cyclin E
expression. Neutralizing A4.1 antibody failed to inhibit cell proliferation in embryonic
fibroblasts derived from p21-deficient mice. These results suggest that cell cycle arrest in cells
treated with neutralizing A4.1 antibody results, at least in part, from p21-dependent inhibition of
cdk2 function. Taken together, these data implicate p21 in TSP1-dependent regulation of cellular
growth. Future studies should elucidate the molecular mechanisms underlying A4.1-dependent
regulation of p21 expression.
Acknowledgements
We are grateful to Dr. P. Leder for the gift of wild-type and p21-deficient MEFS, and to
Dr. P. Bornstein for critical reading of the manuscript. Supported in part by National Institutes of
Health Grants HL 57519 and AG 15227 (V. A.); HL 40518, HL 53354 and HL 57516 (J. M. I.);
CA 65872 (W. A. F.).
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Figure Legends
Figure 1. Antibody blockade of TSP1 inhibits serum-inducible VSMC proliferation. (A)
Serum-starved VSMCs (black bar) were stimulated with DMEM supplemented with 10% FBS
and control mouse IgM (100 g/ml, stripped bar) or with the indicated concentrations of A4.1
anti-TSP1 antibody. After 18 h, [3H]-thymidine was added to the medium (3 Ci/ml) and cells
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3
were incubated for an additional 6 h. [ H]-thymidine uptake was determined in TCA precipitates.
Each bar represents the mean ± SEM of three independent experiments. The asterisks indicate
statistically significant differences in A4.1-treated cells versus cells treated with control mouse
IgM (p<0.01). (B). A4.1 antibody blocked S-phase entry in VSMCs stimulated by serum.
Starvation-synchronized VSMCs were switched to growth medium (10% FBS) supplemented
with either control IgM or A4.1 antibody (100g/ml). After 24 h, cells were washed with PBS
0
and harvested by trypsinization. Cells were then fixed in 70% ethanol overnight at 4 C and
stained with propidium iodide. DNA content was analyzed by flow cytometry to determine the
cell cycle distribution. Each bar represents the mean ± SEM of three independent experiments.
Figure 2. A4.1 antibody blocks serum-inducible cdk2-dependent kinase activity. Cell
extracts were immunoprecipitated with anti-cdk2 antibodies and the kinase activity in the
immunopellets was assayed by using histone H1 and -[32P]ATP as substrates. Reaction mixtures
were separated onto 12% SDS-PAGE, and the gels were dried and autoradiographed. The
arrowheads point to phosphorylated histone H1. (A) C2C12 skeletal muscle cells (SKMC) and
VSMCs were maintained in growth medium (P=proliferating) or were serum–starved for 3 d (Q).
Parallel dishes were also serum-restimulated for 18 h following starvation (Q+FBS). (B) Cell
extracts were prepared from starvation-synchronized VSMCs (0.2% FBS, lane 1), and from cells
that had been serum-restimulated for 16 h with growth medium after serum starvation (10 %
FBS, lanes 2-4). Serum-stimulated cells were treated with control mouse IgM (lane 3) or A4.1
anti-TSP1 antibody (lane 4) (100 g/ml).
Figure 3. Antibody blockade of TSP1 inhibits serum-inducible cyclin A and cyclin E
expression and induces p21 protein levels in VSMCs. Cell extracts (50 g protein) prepared from
VSMCs treated as indicated in Fig. 2B were subjected to Western blot analysis using anti-cdk2,
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anti-cyclin E, anti-cyclin A, anti-p27 and anti-p21 antibodies. Densitometric analysis was
performed to quantify the relative amount of protein in each band. Results are shown below each
lane and are expressed relative to the amount in serum-starved cells (lane 1 = 100%). To
determine the amount of p21 bound to cdk2, 200 g of cell extract was immunoprecipitated with
anti-cdk2 antibodies. The immunopellets were resuspended in sample loading buffer and
separated by electrophoresis on 12% SDS-PAGE. After transfer, the blot was subjected to
Western blot analysis using anti-p21 antibodies.
Figure 4. p21 is essential for A4.1-dependent cell cycle arrest in mouse embryonic fibroblasts.
MEFS were isolated from wild-type (A) and p21-deficient (B) mice. Cells were serum-starved by
incubation in 0.2% FBS for 72 hours. Cultures were switched to growth medium (10% FBS)
supplemented with either control IgM (100 g/ml, black bar) or with the indicated amount of
A4.1 antibody. [3H]-thymidine was added to the culture medium 12 h post-serum stimulation,
cells were incubated for an additional 4 h and incorporated label was determined by TCA
precipitation. The results represent the mean ± SEM of three experiments.
23