Distinct regulation of mitogen-activated protein kinases and p27Kip1 in smooth muscle
cells from different vascular beds: A potential role in establishing regional phenotypic
variance.
Claudia Castro, Antonio Díez-Juan, María José Cortés* and Vicente Andrés**
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and
Therapy, Instituto de Biomedicina de Valencia (IBV-CSIC), Spanish Council for Scientific
Research, 46010-Valencia, Spain
* Present address: Departments of Medicine and Biology, University of California, San Diego,
La Jolla, California.
**Author for correspondence:
Instituto de Biomedicina de Valencia (IBV-CSIC)
C/ Jaime Roig, 11
46010 Valencia (Spain)
Telephone: 34-96-3391752
FAX: 34-96-3690800
Email: vandres@ibv.csic.es
RUNNING TITLE: Regional control of SMC phenotype by MAPKs and p27
KEY WORDS: smooth muscle cell, proliferation, migration, p27, MAPK, cardiovascular
disease.
1
SUMMARY
Excessive proliferation and migration of vascular smooth muscle cells (SMCs)
participate in atherosclerotic plaque growth. In this study, we investigated whether SMCs from
vessels with different atherogenicity exhibit distinct growth and migratory potential, and
investigated the underlying mechanisms. In fat-fed rabbits, we found increased cell
proliferation and atheroma formation in the aortic arch versus the femoral artery. When
examined in culture, SMCs isolated from the aortic arch (ASMCs) displayed a greater capacity
for inducible proliferation and migration than paired cultures of femoral artery SMCs (FSMCs).
Two lines of evidence suggested that distinct regulation of the growth suppressor p27 Kip1 (p27)
contributes to establishing these phenotypic dissimilarities. First, p27 expression was
comparably lower in ASMCs, which exhibited a higher fraction of p27 phosphorylated on
threonine 187 (Thr187) and ubiquitinated. Second, forced p27 overexpression in ASMCs
impaired their proliferative and migratory potential. We found that PDGF-BB-dependent
induction of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase
(ERK) pathway was comparably higher in ASMCs. Importantly, pharmacological inhibition of
MAPKs increased p27 expression and attenuated ASMC proliferation and migration. In
contrast, forced MAPK activation diminished p27 expression and markedly augmented FSMC
proliferation and migration. We propose that intrinsic differences in the regulation of MAPKs
and p27 play an important role in creating variance in the proliferative and migratory capacity
of vascular SMCs, which might in turn contribute to establishing regional variability in
atherogenicity.
2
INTRODUCTION
Atherosclerotic cardiovascular disease is the leading cause of mortality and morbidity in
developed countries. Although percutaneous transluminal angioplasty has become a wellestablished technique for revascularization of patients with arterial occlusive disease, the
occurrence of restenosis at the site of angioplasty remains the major limitation despite a
successful procedure. The molecular basis of atherosclerosis and restenosis involves
dedifferentiation of vascular SMCs to a socalled “synthetic state” characterized by abundant
production of matrix components and excessive proliferative and migratory activities (1-3).
Therefore, a better understanding of the molecular mechanisms underlying these processes
should help develop novel therapeutic approaches for the treatment of cardiovascular disease.
Cellular proliferation is regulated by the balance between multiple cyclin-dependent
kinase (CDK)/cyclin holoenzymes and members of the Cip/Kip and INK4 families of CDK
inhibitors (CKIs) (4,5). Active CDK/cyclin complexes promote cell cycle progression by
phosphorylating the retinoblastoma gene product (pRb) and the related pocket proteins p107
and p130 from mid G1 to mitosis. CKIs associate with and inhibit the activity of CDK/cyclin
holoenzymes. Studies arguing for a role of the Cip/Kip protein p27 in the pathophysiology of
the cardiovascular system include the following: 1) p27 may contribute to the reestablishment
of the quiescent phenotype after the initial proliferative response to balloon angioplasty in rat
and porcine arteries, and adenovirus-mediated overexpression of p27 inhibited neointimal
growth in these experimental models (6-8); 2) p27 may function as a molecular switch that
regulates the phenotypic response of vascular SMCs to both hyperplastic and hypertrophic
stimuli (9,10); 3) p27 is a negative regulator of endothelial cell proliferation and migration in
vitro, and adenovirus-mediated overexpression of p27 inhibited angiogenesis in vivo (11,12);
4) p27 may contribute to integrin-mediated control of vascular SMC proliferation (13); 5) p27
may limit cardiomyocyte proliferation during early postnatal development and after injury in
3
adult mice (14,15); 6) changes in p27 expression might regulate human vascular cell
proliferation within atherosclerotic lesions (7,16), and a causal link between reduced p27
expression and atherosclerosis has been established in apolipoprotein E-deficient mice (17). It
has been established that expression of p27 is regulated mainly at the level of translation and
protein turnover (18).
Multiple growth factors and cytokines interact with specific receptors located in the
cytoplasmic membrane of vascular cells in response to a variety of pathological stimuli, thus
triggering a complex signal transduction cascade which culminates in changes in gene
expression that execute a proliferative and migratory response (2,3). Activation of the MAPK
signal transduction pathway is thought to play an important role during cardiovascular disease
(19-23).
It has been well established that different segments of the arterial tree display
significant differences in their susceptibility to atherosclerosis, both in animal models and
humans. In this regard, it is notable that vascular SMCs display regional phenotypic variance,
both when comparing cells obtained from different compartments of the same vessel or cells
isolated from vessels from different vascular beds (24-30). The findings of the present study
demonstrate that p27 and MAPKs are critical regulators of vascular SMC proliferation and
migration. Our results suggest that intrinsic differences in the regulation of p27 and MAPKs
may contribute to the establishment of regional variance in the proliferative and migratory
capacity of SMCs from distinct regions of the vascular system.
4
EXPERIMENTAL PROCEDURES
Antibodies
The following antibodies were purchased from Santa Cruz Biotechnology: cyclin D1
(sc-450), cyclin A (sc-751), cyclin E (sc-198), p27 (sc-1641), -tubulin (sc-8035), CDK2 (sc163-G), PDGFR- (sc-432), p-ERK1/2 (sc-7383, reactive with Tyr-204 phosphorylated ERK1
and ERK2), ERK2 (sc-154, reactive with ERK2 and, to a lesser extent, ERK1). Other
antibodies were purchased from Calbiochem (anti-p27 phospho-specific Thr187, reference
506128, and anti-ubiquitin, reference 662099), Dako (anti-5-bromodeoxyuridine), and Master
Diagnostica (anti-smooth muscle  actin, clone 1A4, and anti-desmin, clone ZC18)
Rabbit studies
Male white New Zealand rabbits (4-5-month-old) were fed either control chow (n = 5)
or received for 2 months a high-fat diet (n = 10) containing 10 g of cholesterol (Sigma) and 60
ml of peanut oil per kilogram of control chow (1% cholesterol). Animals received 4
intraperitoneal injections of 5-bromodeoxyuridine (BrdU) (Sigma, 20 mg/Kg each) at 12-hour
intervals starting 48 h before sacrifice. Rabbits were killed with an overdose of pentobarbital. A
cut was made in the cava vein and the systemic circulation was thoroughly perfused with saline
through the heart. The aortic arch and the right femoral artery were fixed in situ with 100%
methanol. Arteries were removed, fixation was continued overnight and tissues were paraffinembedded and cut in 5 m cross-sections. Immunohistochemistry using mouse monoclonal
anti-BrdU antibody (1/50) was done with a biotin/streptavidin-peroxidase detection system
(Signet Laboratories) and DAB substrate (Sigma).
Cell culture and retroviral infection
The aortic arch, the common carotid artery and the femoral artery of 4-month-old male
New Zealand white rabbits were extracted to prepare primary cultures (ASMCs, CSMCs and
5
FSMCs, respectively). Arteries were dissected free from surrounding tissue and adventitia and
cut into small pieces. Aortic tissue was digested with collagenase (2 mg/mL, Worthington) in
DMEM-F12 supplemented with 5% FBS for 3 h in a shaking bath at 37C. Cells were
incubated at 37C in a humidified 5% CO2-95% O2 atmosphere in DMEM-F12 supplemented
with 10% FCS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L L-glutamine. All
studies were carried out with primary cultures between passages 2 and 8. Pharmacological
inhibition of MAPK kinase (MEK) was achieved by exposing ASMC cultures to PD98059
(Tocris) as indicated in figure legends.
Recombinant retrovirus were generated using the retroviral vectors pBabePuro-p27wt
(31) and pBabePuro-MEKE, which encode for wild-type p27 and a constitutively active MEK1
mutant (32), respectively. pBabePuro-MEKE was generated by digesting pcDNAIII-MEKE
(gift of C. Caelles) with BamHI and XhoI and subcloning the MEKE cDNA into pBabePuro.
Infection of asynchronously growing cells was performed as suggested by the supplier of the
PT67 packaging cells (Clontech). Infected cells were selected in the presence of puromycin
(2.5 g/mL) (Sigma).
Immunofluorescence labeling of vascular SMC differentiation markers and TUNEL
assay.
Cells were plated onto glass coverslips. To examine the expression of differentiation
markers, cells were grown until reaching confluence and then were maintained in mitogen-free
ITC media (33) for 2 days. Cells were fixed with 4% paraformaldehyde in PBS at room
temperature for 1 hr and permeabilized with 0.1% Triton X-100/PBS. Cells were blocked with
1% BSA/PBS and expression of smooth muscle -actin (SM -actin) and desmin was
examined by indirect immunofluorescence. Microscopic images were digitally recorded on an
Axioscope II microscope (Zeiss).
6
For TUNEL assays, cells were grown to ~60% confluence and were maintained in
mitogen-free ITC media (Invitrogen) for 2 days. For ultraviolet (UV) light irradiation, cell
culture media was removed and the cells were washed twice with PBS. Then, cultures were
placed in the tissue culture hood and exposed to UV light for 45 minutes (UV G-30 Watt lamp,
Sylvania, Japan). Control (not irradiated) and UV-irradiated cells were fixed and permeabilized
as indicated above, and TUNEL assay was performed using an in situ cell death detection kit as
suggested by the manufacturer (Boehring Mannheim, Mannheim, Germany).
Proliferation assays
Cells for 3H-thymidine incorporation assays were plated in 10% FBS/DMEM-F12 at a
density of 4x104 cells/well in 12-well plates. When ~80% confluence was reached, cells were
rendered quiescent by maintaining cultures for 48-72 hours in mitogen-free ITC media (33).
Starvation-synchronized cultures were stimulated with PDGF-BB (10 ng/mL) to induce cell
cycle reentry and cells were pulsed with 1 mCi/L 3H-thymidine (Amersham) during the last 4 h
of incubation. After washes with cold PBS, DNA was precipitated with 15% trichloroacetic
acid and solubilized with 0.2 mol/L NaOH. Radioactivity incorporated into DNA was measured
in a scintillation counter (Wallac).
Migration assays
Migration of cultured cells labeled with the fluorescent dye Calcein-AM (Molecular
Probes) was assessed with the FALCON HTS FluoroBlock system as suggested by the
manufacturer (Becton Dickinson, Bedford). Labeled cells were placed in the inserts (8.0 m
pore size, 5x104 cells/insert) in serum-free media. The lower chamber contained either serumfree media (unstimulated cells) or the chemotactic agent (10% FBS or 10 ng/mL PDGF-BB)
(induced cells). Serum-free media was supplemented with 0.1% BSA. Chemotaxis at different
times after plating the cells was assessed by detecting the fluorescence in the lower chamber
7
using a Victor 4120 multilabel counter (Wallac). Results represent the average fluorescence of
induced cells (n = 3) after subtracting the fluorescence of unstimulated cells (n = 2-3).
Western blot analysis, immunoprecipitation and CDK assays
Cell lysates were prepared with either ice-cold lysis buffer A or buffer B supplemented
with protease inhibitor Complete Mini cocktail (Roche). Buffer A contained 50 mmol/L Hepes
[pH 7.5], 1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L DTT, 0.1 mM orthovanadate, 10 mM
-glicerophosphate and 10mM sodium fluoride. Buffer B contained 20 mmol/L Tris-HCl [pH
7.5], 0.5% Triton X-100, 0.5% deoxycholate, 150 mmol/L NaCl, 10 mmol/L EDTA, 1 mmol/L
DTT. Fifty g of protein was electrophoresed on 12% SDS-PAGE to perform Western blot
analysis as described previously (6). Antibody dilutions were 1:100 (cyclin D1, cyclin A, cyclin
E, p-ERK1/2, p27), 1:200 (-tubulin, CDK2), 1:250 (PDGFR-), 1:500 (anti-p27 phosphospecific Thr187) and 1:700 (ERK2). For immunoprecipitation/Western blot assays, cell lysates
were incubated with anti-ubiquitin antibody (0.5 g) and protein A/G Plus-agarose (Santa Cruz
Biotechnologies) for 4 at 4 °C under rotation. The immune complexes were extensively washed
and subjected to Western blot analysis using anti-p27 antibody.
CDK activity in cell lysates (100 µg protein) was determined as previously described
(6), except that CDK/cyclin holoenzymes were immunoprecipitated with 0.2 µg of each anticyclin E and anti-cyclin A antibodies.
Statistical analysis
Results are reported as mean  SEM. Differences were evaluated using either two-tail,
unpaired Student’s t test, or ANOVA and Fisher’s post-hoc test (Statview, SAS institute).
8
RESULTS
Arterial
cell
proliferation
and
atherogenesis
in
different
vascular
beds
of
hypercholesterolemic rabbits
We investigated atherogenesis in fat-fed New Zealand white rabbits, which rapidly
develop atheromas in response to dietary manipulation (34). To examine arterial cell
proliferation, animals received 4 injections of BrdU prior to sacrifice. While aortic
atherosclerosis and BrdU immunoreactivity were essentially undetectable in rabbits fed control
chow (n = 5, data not shown), all of the fat-fed rabbits included in our study displayed
atheromatous lesions in the aortic arch and exhibited abundant BrdU immunoreactivity in both
intimal and medial cells (n = 10, Fig. 1A). In marked contrast, only 3 of 10 fat-fed rabbits
displayed small atherosclerotic lesions in the femoral artery (Fig. 1B). Moreover, the number of
BrdU-positive cells in femoral arteries was negligible in the media and was lower within the
lesions as compared to the aortic arch (Fig. 1B). These findings are consistent with previous
rabbit studies demonstrating that the aortic arch is highly susceptible to diet-induced
atherosclerosis (34-37).
ASMCs and FSMCs display dissimilar migratory and proliferative activity in vitro
Having demonstrated distinct proliferative response and atherogenicity in the aortic arch
and femoral artery, we isolated SMCs from these vessels (ASMCs and FSMCs, respectively) to
ascertain whether their phenotypic dissimilarities were maintained in vitro. In primary cultures
grown to confluence in serum-free media, ASMCs exhibited an epithelioid shape (Fig. 2A),
whereas FSMCs disclosed a bipolar, spindle-shaped morphology (Fig. 2B). We next performed
indirect immunofluorescence experiments in passage 2 cultures to examine the expression of
SMC differentiation markers. Both ASMCs and FSMCs revealed abundant SM -actin
immunoreactivity in a prominent stress fiber pattern (Fig. 2C, D). In contrast, desmin
9
expression appeared more abundant in FSMCs (Fig. 2E,F). These phenotypes were stable at
least up to passage 8 (data not shown).
We next compared the migratory and proliferative capacity of cultured ASMCs and
FSMCs. While FSMCs did not migrate in response to 6 hours of stimulation with either PDGFBB or FBS, both agents elicited a robust migratory response in paired cultures of ASMCs (Fig.
2G). Likewise, 3H-thymidine incorporation in starvation-synchronized cultures restimulated
with PDGF-BB was lower in FSMCs (Fig. 2H). For example, compared with starved cultures,
maximum 3H-thymidine incorporation at 24 h post-stimulation increased by 16- and 42-fold in
FSMCs and ASMCs, respectively. The proliferative response toward 10% FBS was also
stronger in ASMCs (data not shown). In contrast, as determined by the TUNEL assay,
apoptosis was similar in ASMCs and FSMCs, both under control conditions and after UV
irradiation (Fig. 2I).
Lineage analysis experiments have suggested that neural crest-derived (ectoderm) SMCs
prevail in arterial segments proximal to the heart (i. e., aortic arch and great vessels of the head
and neck), whereas arteries located more distally to the heart contain mainly mesoderm-derived
SMCs (i.e., abdominal aorta and hindlimb arteries) (1,27,38). Thus, dissimilar behavior and
morphology of ASMCs and FSMCs raised the possibility that adult SMC phenotypic properties
are related, at least in part, to their primary embryonic lineage. Consistent with this notion, we
found that carotid artery SMCs (CSMCs) (also of neural crest origin) behaved in a similar
fashion as the ASMCs in proliferation and migration assays (Fig. 3).
Role of p27 in the establishment of phenotypic variance between ASMCs and FSMCs
Differences in proliferation and migration between ASMCs and FSMCs prompted us to
investigate the underlying molecular mechanisms. Consistent with the results of Fig. 2H
showing greater PDGF-BB-dependent proliferation in ASMCs than in FSMCs, CDK activity
was higher in PDGF-BB-stimulated ASMCs (Fig. 4A). Likewise, upregulation of the positive
10
cell cycle regulators cyclin D1 and cyclin A, whose expression is induced as starvationsynchronized cells resume progression through G1 and S-phase upon mitogen restimulation
(4,5), occurred earlier and was more prominent in PDGF-BB-stimulated ASMCs versus
FSMCs (Fig. 4B). Expression of the PDGF receptor isoform  (PDGFR-) was similar in
ASMCs and FSMCs, both under mitogen-free conditions and upon PDGF-BB stimulation (Fig.
4C), suggesting that dissimilar PDGF-BB-dependent proliferation and migration in ASMCs
and FSMCs was not a consequence of distinct regulation of PDGFR- expression.
Downregulation of PDGFR- 9 hours after PDGF-BB stimulation is consistent with the notion
that binding of PDGF to its receptor leads to internalization and degradation of the ligandreceptor complex in endosomes (39).
We next investigated the expression of the growth suppressor p27 in the same confluent
cultures of ASMC and FSMC used for the PDGFR- immunoblot. Of note, the lysis buffer
used in these assays did not contain phosphatase inhibitors (buffer B). Both under mitogen-free
conditions and at different time points after PDGF-BB stimulation, p27 was detected as a single
band that was more abundant in confluent cultures of FSMCs versus ASMCs (Fig. 4C). For
example, while p27 was not detected in ASMC after 9 hours of stimulation, FSMCs expressed
at this time point more p27 than did unstimulated ASMCs. Analysis of subconfluent cultures
also disclosed higher level of p27 expression in FSMCs (data not shown). We next examined
cell lysates prepared in the presence of phosphatase inhibitors (buffer A), which also disclosed
higher p27 expression in FSMCs versus ASMC (Fig. 4D, top blot). Notably, these experiments
demonstrated the presence of two p27 immunoreactive bands of different electrophoretic
mobility and distinct relative abundance in these cells. Averaged over four experiments, the
slower migrating band (open arrowhead) predominated in ASMCs (89.7 % ± 8.0), whereas the
faster migrating band (closed arrowhead) prevailed in FSMCs (95.7 % ± 1.5). Western blot
analysis using a phospho-specific antibody identified the slower migrating band as p27
11
phosphorylated on Thr187 (Fig. 4D, middle blot). This phosphorylation event is thought to
initiate the major pathway for p27 proteolysis via a mechanism involving its ubiquitination and
subsequent turnover in the proteasome (18). Consistent with this notion, immunoprecipitation
experiments using an anti-ubiquitin antibody followed by Western blot analysis revealed the
presence of ubiquitinated p27 in the slower migrating p27 immunoreactive band in both
ASMCs and FSMCs (Fig. 4E). It is noteworthy that the faster migrating p27 immnuoreactive
band in ASMCs, but not in FSMCs, also contained ubiquitinated p27 (see Discussion).
Collectively, these results suggest that the majority of p27 in ASMCs undergoes
phosphorylation on Thr187 and ubiquitination, whereas these posttranslational modifications
are detected only in a small fraction of p27 in FSMCs.
We next investigated the effect of p27 overexpression on ASMC proliferation and
migration by infecting these cells with retroviral vectors encoding for p27 (Rev-p27). Rev-p27infected ASMCs disclosed a 3-fold increase in p27 expression, which caused a reduction in 3Hthymidine incorporation (Fig. 5A) and migration (Fig. 5B) as compared with control cultures
infected with Rev-LacZ. These findings demonstrate that increased p27 expression is sufficient
to attenuate the growth and migratory capacity of ASMCs. Thus, distinct regulation of p27
expression might contribute to establishing differences in the proliferative and migratory
capacity of ASMCs and FSMCs.
Differential regulation of MAPKs in ASMCs and FSMCs and role in the regulation of
vascular SMC proliferation and migration
As the MAPK pathway plays a pivotal role in transducing environmental signals
required for both cellular proliferation and migration (40), we examined the kinetics of
expression and activation of individual MAPKs in ASMCs and FSMCs. Western blot analysis
using an antibody specific for the phosphorylated (active) form of the MAPK isoforms of 44
and 42 kDa (dubbed extracellular signal-regulated kinases, ERK1 and ERK2, respectively)
12
revealed a rapid activation of these proteins upon PDGF-BB stimulation of mitogen-depleted
ASMCs and FSMCs (Fig. 6, top blot). However, maximum level of ERK1/2 activation was
higher in ASMCs than in FSMCs. Moreover, ERK1/2 activation was more prolonged in
ASMCs. These differences occurred in spite of similar level of total ERK1/2 in ASMCs and
FSMCs (Fig. 6, bottom blot).
To determine whether dissimilar MAPK regulation might contribute to phenotypic
differences between ASMCs and FSMCs, we performed loss- and gain-of-function
experiments. Treatment of ASMCs with PD98059, a selective inhibitor of MEK, impaired
PDGF-BB-dependent ERK1/2 activation (Fig. 7A) and upregulated p27 expression (Fig. 7B).
Importantly, exposure of asynchronously growing ASMCs to PD98059 inhibited 3H-thymidine
incorporation in a dose-dependent manner (Fig. 7C), and preincubation of starvationsynchronized ASMCs with PD98059 blocked de novo DNA synthesis upon mitogen
stimulation (Fig. 7D). Moreover, exposure of ASMCs to PD98059 inhibited migration (Fig.
8A).
We also examined the effect of forced ERK1/2 activation on FSMC proliferation and
migration by infecting cultures with a retroviral vector encoding for a constitutively active
MEK1 mutant (Rev-MEKE). As compared to control cultures, Rev-MEKE-infected FSMCs
disclosed constitutive activation of ERK1/2 (Fig. 9A), which markedly reduced p27 expression
(Fig. 9B), increased 3H-thymidine incorporation (Fig. 9C) and augmented cell migration (Fig.
8B). Collectively, the above studies suggest that differential regulation of ERK1/2 in ASMCs
and FSMCs plays an important role in the establishment of intrinsic differences in the
proliferative and migratory potential of these cells.
13
DISCUSSION
Vascular SMCs undergo dedifferentiation and excessive proliferation and migration
during atherosclerosis and restenosis post-angioplasty (1-3). Upregulation of the growth
suppressor p27 in the arterial wall might limit SMC proliferation at late time points after
balloon angioplasty in rat and porcine arteries (6,7), and adenovirus-mediated overexpression
of p27 inhibited neointimal thickening in these animal models (8,41). Regarding the role of p27
on atherosclerosis, genetic disruption of p27 increased arterial cell proliferation and accelerated
atheroma formation in hypercholesterolemic apolipoprotein E-deficient mice (17). Moreover,
p27 might mediate TGF-dependent inhibition of cell growth in human atheromas (16), and
proliferating cells within human coronary atheromas appear to express low level of p27 (7).
Consistent with the observation that p27 overexpression attenuated human vascular endothelial
cell migration in vitro (12), and that p27 inactivation reduced rapamycin-dependent inhibition
of vascular SMC migration (42), we found that retrovirus-mediated overexpression of p27
inhibited vascular SMC migration. Thus, p27 might control neointimal thickening via
regulation of both cell proliferation and migration.
Our studies with fat-fed rabbits showed that aortic arch tissue displays increased cell
proliferation and atherogenicity as compared to femoral artery. We found that primary cultures
of ASMCs and FSMCs maintained marked differences in their growth and migratory potential,
which might be related, at least in part, to their distinct primary embryonic lineage (neural crest
and mesoderm, respectively) (1,27,38). Indeed, ASMCs and CSMCs, which are thought to
derive from neural crest ectoderm, behaved similarly in our proliferation and migration assays.
We chose to examine ASMCs and FSMCs as an in vitro model to elucidate molecular
mechanisms involved in the establishment of dissimilar atherogenicity in distinct vessel
segments. Greater ASMC proliferation and migration correlated with lower expression of p27
when compared to FSMCs, and retrovirus-mediated overexpression of p27 attenuated the
14
growth and migratory potential of ASMCs. Previous studies also support the notion that distinct
regulation of p27 expression plays an important role in establishing differences in the
phenotypic response of vascular SMCs toward a variety of stimuli. First, Yang et al. (29)
reported reduced proliferation of human internal mammary artery (IMA) compared with
saphenous vein (SV) SMCs. Importantly, PDGF-BB markedly downregulated p27 protein level
in SV, but this response was much less pronounced in IMA. Thus, sustained p27 expression in
spite of growth stimuli may contribute to the resistance to growth of SMCs from IMA, and to
the longer patency of arterial versus venous grafts. Second, p27 may regulate the proliferative
response of vascular SMCs toward fibroblast growth factor 2 (FGF2 or basic FGF). While
FGF2 plays a critical role in the induction of medial SMC proliferation after balloon
angioplasty (30,43,44), neutralizing antibodies to FGF2 failed to inhibit neointimal SMC
proliferation in balloon-injured arteries (45). Moreover, only a small increase in growth was
observed when arteries with existing neointimal lesions were esposed to FGF2 (30,43).
Attenuated FGF2-dependent proliferation of neointimal SMCs occurred despite a robust
induction of positive cell cycle regulators (30). Interestingly, neointimal SMCs expressed high
levels of p27 compared with medial SMCs, and FGF2 infusion did not reduce the level of this
inhibitor in arteries with established neointimal lesions.
Protein turnover is thought to play a major role in the regulation of p27 expression.
Phosphorylation of p27 on Thr187 triggers its ubiquitination and rapid turnover in the
proteasome (18). Our Western blot assays demonstrate that the majority (90 %) of p27 in
ASMCs corresponds to a slow migrating form that undergoes phosphorylation on Thr187 and
ubiquitination. In marked contrast, approximately 96% of p27 in FSMCs corresponded to a
faster migrating p27 band that was not recognized by the phospho-specific antibody and did not
contain ubiquininated protein. Thus, the relative amount of p27 phosphorylated on Thr187 and
ubiquitinated appears higher in ASMCs compared to FSMCs, which might account for the
15
lower level of p27 detected in ASMCs. Of note, ubiquitinated p27 in the faster migrating band
that does not contain phosphorylated Thr187 was also detected in ASMCs (cf. Fig. 4E). This
finding is in agreement with recent studies demonstrating an additional pathway for p27
ubiquitination and proteolysis independent of phosphorylation of p27 on Thr187 (46,47).
We investigated additional regulatory networks involved in the establishment of
vascular SMC phenotypic variance. A wealth of evidence implicates the rapid activation of the
MAPK signal transduction pathway during the pathogenesis of cardiovascular disease (19,21).
For example, it has been suggested that persistent activation and hyperexpression of ERK1/2 is
a critical element to initiate and perpetuate cell proliferation during diet-induced atherogenesis
in the rabbit (48). Moreover, ERK1/2 activation occurs rapidly after angioplasty of porcine and
rat arteries, (20,22), and all three MAPKs are activated in human failing hearts (49). Our results
indicate that ERK1/2 contribute to establishing phenotypic differences between ASMCs and
FSMCs. First, mitogen-dependent activation of ERK1/2 was more robust in ASMCs than in
FSMCs. Second, reduced ERK1/2 activation by exposure of ASMCs to PD98059 impaired
their growth and migratory capacity. By contrast, forced activation of ERK1/2 greatly increased
FSMC proliferation and migration. We observed increased p27 expression upon ERK1/2
blockade in ASMCs, and diminished p27 expression upon forced ERK1/2 activation in FSMCs.
Thus, in agreement with previous studies in NIH 3T3 fibroblasts and cancer cells (50-53), our
findings suggest an important role for the MAPK pathway in the control of p27 expression in
ASMCs and FSMCs. Solid ERK1/2 activation in mitogen-stimulated ASMC cultures might
facilitate p27 degradation, thus favoring proliferation and migration of these cells. In contrast,
weaker ERK1/2 activation might contribute to comparably higher expression of p27 in FSMCs,
thus hindering their proliferative and migratory responses. In consideration of this model, it is
noteworthy that PDGF-BB induced similar MAPK activation in cultures of SV and IMA in
16
spite of distinct regulation of p27 in these cells (29), suggesting that MAPK-independent
mechanisms of p27 regulation might operate in SMCs of different vascular beds.
In conclusion, we propose that intrinsic differences in MAPK-dependent signaling and
p27 expression in rabbit ASMCs and FSMCs contribute to establishing variance in their
proliferative and migratory potential. These dissimilarities might be attributable, at least in part,
to their distinct primary embryonic origin. Further clarification of the molecular networks
underlying vascular SMC phenotypic variance should shed significant insight into the
mechanisms leading to regional variability in the susceptibility to intimal lesion development.
17
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21
FOOTNOTES
1) This work is dedicated to Dr. Jeffrey M. Isner
2) Acknowledgments: We thank C. Caelles for providing pcDNAIII-MEKE. This work was
supported by grants from the Spanish Government and Fondo Europeo de Desarrollo Regional
(PM97-0136, SAF2001-2358). C. Castro received salary support from Agencia Española de
Cooperación Internacional (AECI). A. Díez-Juan was partially supported from the Spanish
DGESIC and Fondo Europeo de Desarrollo Regional (grant 1FD97-1035-C02-02), and from
Fondo Social Europeo (CSIC-Programa I3P fellowship).
22
FIGURE LEGENDS
Fig. 1: Arterial cell proliferation and atherogenesis in the arotic arch and femoral
artery of hypercholesterolemic rabbits. Rabbits received either control chow (n = 5) or a
high-fat diet (n = 10) for 2 months. Prior to sacrifice, animals were injected with BrdU to assess
arterial cell proliferation. The photomicrographs show representative examples of BrdU
immunoreactivity in cross-sections of the aortic arch (A) and femoral arteries (B) of fat-fed
rabbits. Specimens were counterstained with eosin. Two different magnifications are shown for
each specimen as indicated in each photomicrograph. Arrows in the 200x photomicrograph of
the femoral artery indicate two BrdU-positive cells within the intimal lesion. White arrowheads
point to the internal elastic lamina.
Fig. 2: Phenotypic differences between ASMCs and FSMCs. Phase contrast
microscopic view of primary cultures of ASMCs (A) and FSMCs (B) (200x magnification). (CF) Indirect immunofluorescence analysis of passage 2 primary cultures of ASMCs (C,E) and
FSMCs (D,F) (400x magnification). ASMCs and FSMCs disclosed similar expression of SMactin (C,D). By contrast desmin expression was low in ASMCs (E) and high in FSMCs (F).
The right panel in E and F shows nuclear staining (Hoechst 33258) in the same field shown for
desmin staining (left). Phenotypic differences between ASMCs and FSMCs were maintained
up to passage 8. (G) Primary cultures (passage 6) were labeled with the fluorescent dye Calcein
AM and were placed in serum-free media in the upper chamber of FALCON HTS FluoroBlock
inserts. The lower chamber contained either serum-free media, 10 ng/mL PDGF-BB (upper
panel) or 10% FBS (lower panel). Chemotaxis was assessed by detecting the fluorescence of
cells migrating to the lower chamber at the indicated time points after plating the cells. Results
represent the average fluorescence of PDGF-BB-induced or 10% FBS stimulated cells after
subtracting the fluorescence of unstimulated cells (n = 3). Differences were evaluated using
ANOVA and Fisher’s post hoc test. Only comparisons versus t = 0 are shown. * p < 0.05, **, p
23
< 0.005, and ***, p < 0.0001. (H) Cells were maintained for 72 h in mitogen-free ITC media
and then were exposed to 10 ng/mL PDGF-BB for the indicated time. Cultures were pulsed
with 3H-thymidine. Results represent the average of 3 experiments using passage 3, 4 and 6
cultures. Differences were evaluated using ANOVA and Fisher’s post hoc test. Comparisons
versus t = 0: *, p < 0.025, **, p < 0.015, ***, p < 0.0001; comparisons between ASMC and
FSMC at each time point: †, p < 0.0001 (n = 6 each time point). (I) Percentage of TUNELpositive cells in starvation-synchronized cultures. Analysis included control and UV-irradiated
cells. The total number of cells analyzed in 10 high-power fields (400x) is indicated below each
bar.
Fig. 3: ASMCs and CSMCs display similar migratory and proliferative capacity.
Statistical analysis was performed using ANOVA and Fisher’s post hoc test. (A) Migration was
assayed as described in Fig. 2G using 10% FBS as the chemotactic agent. *, p < 0.0001 (versus
t = 0). (B) 3H-thymidine incorporation was assayed as indicated in Fig. 2H (n = 4 each time
point). *, p < 0.0001 (versus t = 0); †, p < 0.001 (comparisons between ASMC and CSMC at
each time point).
Fig. 4: ASMCs and FSMCs display dissimilar cell cycle regulatory protein
expression and CDK activation. Confluent cultures were maintained for 72 h in mitogen-free
ITC media and then exposed to 10 ng/mL PDGF-BB as indicated. Cell extracts were prepared
in lysis buffer A containing phosphatase inhibitors (A,B,D,E) or buffer B (C), which did not
contain phosphatase inhibitors. Analysis of lysates included cyclin A/cyclin E-associated CDK
activity using histone H1 and -32P-ATP substrates (A), Western blot with the indicated
antibodies (B,C,D), and immunoprecipitation with an anti-ubiquitin antibody followed by
Western blot of the immunoprecipitated material using anti-p27 antibodies (E). (A) Kinase
reactions were analyzed by SDS-PAGE and autoradiography. Relative activity was estimated
after densitometric analysis (0 hrs is set as 1 for each cell type). (B) Densitometric analysis was
24
performed to estimate the relative p27 level. Each p27 value was divided by its corresponding
tubulin loading control (ASMC at 0 hrs = 1; nd: not detected). (C) Densitometric analysis was
performed to estimate the relative level of cyclin D1 and A. Each cyclin value was divided by
its corresponding CDK2 loading control. Shown below is the PVDF membrane stained with
Ponceau prior to incubation with antibodies. (D, E) The phospho-specific anti-p27 antibody
only recognizes p27 phosphorylated on Thr187. Open and closed arrowheads point to the slow
and faster migrating p27 immunoreactive band, respectively. Note that the slow migrating band
that undergoes phosphorylation on Thr187 and ubiquitinilation prevailed in ASMCs. By
contrast, the faster migrating p27 band that does not contain protein phosphorylated on Thr187
and does not undergo ubiquitination predominated in FSMCs (see text for details).
Fig. 5: Inhibition of ASMC proliferation and migration by retrovirus-mediated p27
overexpression. ASMCs were infected with control retrovirus (Rev-lacZ) or a retrovirus
encoding for p27 (Rev-p27). Infected cells were selected with puromycin. (A) Cells were
maintained in 10% FBS/DMEM, pulsed for 4 h with
3
H-thymidine and radioactivity
incorporated into DNA was quantified. Differences were evaluated using two-tail, unpaired
Student’s t test (*, p < 0.0015, n = 5). Puromycin-resistant cells were also lysed in buffer A to
perform Western blot analysis using anti-p27 and anti--tubulin antibodies. Densitometric
analysis was performed to estimate the relative level of p27. Each p27 value was divided by its
corresponding tubulin loading control (Rev-LacZ = 1). (B) Migration of ASMCs infected with
Rev-LacZ or Rev-p27 was measured as indicated in Fig. 2G using 10% FBS as the chemotactic
agent. Differences were evaluated using ANOVA and Fisher’s post hoc test (comparisons
versus t = 0: *, p < 0.005, **, p < 0.0001; comparisons between Rev-LacZ and Rev-p27 at each
time point: †, p < 0.02, ††, p < 0.0001).
Fig. 6: ASMCs and FSMCs display dissimilar MAPK regulation. Western blot
analysis of cells maintained for 72 h in mitogen-free ITC media and then exposed to 10ng/mL
25
PDGF-BB for the indicated time. Cell lysates were prepared in lysis buffer A. P-ERK1/2 and
ERK1/2 indicate phosphorylated (active) and total ERK1/2, respectively. Densitometric
analysis was performed to estimate the relative level of P-ERK1/2. Each P-ERK value was
divided by its corresponding ERK loading control (ASMC at 0 hrs = 1).
Fig. 7: MAPK inhibition upregulates p27 expression and inhibits PDGF-BBdependent ASMC proliferation. Differences were evaluated using ANOVA and Fisher’s post
hoc test. (A, B) ASMCs were maintained for 72 h in ITC media and then were exposed to 10
ng/mL PDGF-BB for short (A) or long periods of time (8 hrs, B). Cell lysates were prepared in
lysis buffer A to perform immunoblot analysis with the indicated antibodies. Treatment with 50
M PD98059 was initiated 1 h before addition of PDGF-BB. P-ERK1/2 and ERK1/2 indicate
phosphorylated (active) and total ERK1/2, respectively. Densitometric analysis was performed
to estimate the relative level of P-ERK1/2 and p27. Each P-ERK or p27 value was divided by
its corresponding loading control (total ERK or tubulin, respectively; nd: not detected). For
p27, results are shown relative to control (set as 1). (C) Asynchronously growing ASMCs were
treated for 1 h in mitogen-free ITC media supplemented with PD98059 or vehicle, and then
cells were incubated for 24 h with 10 ng/mL PDGF-BB. Cultures were pulsed with 3Hthymidine during the last 4 h (n = 4). Comparisons versus control: *, p < 0.0001; comparisons
versus 2 M PD98059: †, p < 0.04, ††, p < 0.02. (D) Cells were maintained for 72 h in ITC
media and then were exposed to 10 ng/mL PDGF-BB. When indicated, mitogen-depleted
ASMCs were pretreated with 50 M PD98059 for 1 h prior to PDGF-BB stimulation. Cells
were pulsed with 3H-thymidine (n = 4,: *, p < 0.0001 versus control starvation-synchronized
cells; †, p < 0.0001 versus 50 M PD98059).
Fig. 8: Altered MAPK signaling affects ASMC and FSMC migration. Migration
assays were performed as indicated in Fig. 2G using 10 ng/mL PDGF-BB (A) or 10% FBS (B)
as the chemotactic agent. Statistical analysis was done using ANOVA and Fisher’s post hoc
26
test. (A) ASMCs were untreated or exposed to 50 M PD98059 during labeling with calceinAM. PD98059 treatment was maintained in both the upper and lower chamber. Comparisons
versus t = 0: *, p < 0.05, **, p < 0.01; comparisons between control and PD98059 at each time
point: †, p < 0.05, ††, p < 0.02, †††, p < 0.006. (B) Migration of FSMCs infected with RevLacZ or Rev-MEKE. Comparisons versus t = 0: *, p < 0.02, **, p < 0.002, ***, p < 0.0001;
comparisons between Rev-MEKE and Rev-LacZ at each time point: †, p < 0.04, ††, p <
0.0001.
Fig. 9: Forced MAPK activation downregulates p27 expression and stimulates
PDGF-BB-dependent FSMC proliferation. FSMCs were infected with control retrovirus
(Rev-lacZ) or a retrovirus encoding a constitutively active MEK1 mutant (Rev-MEKE).
Infected cells were selected with puromycin. Puromycin-resistant cells were maintained for 72
h in mitogen-free ITC media, then cultures were stimulated with 10 ng/mL PDGF-BB for the
indicated time. (A, B) Cells were lysed in buffer A to perform immunoblot analysis. P-ERK1/2
and ERK1/2 indicate phosphorylated (active) and total ERK1/2, respectively. Densitometric
analysis was performed to estimate the relative level of P-ERK1/2 and p27. Each P-ERK and
p27 value was divided by its corresponding loading control (total ERK and tubulin,
respectively; nd: not detected). For p27, results are shown relative to Rev-LacZ at 0 hrs (set as
1). (C) 3H-thymidine incorporation in mitogen-depleted cells (0 h) and 24 h upon PDGF-BB
stimulation. Differences were evaluated using ANOVA and Fisher’s post hoc test (comparisons
among mitogen-depleted cells: *, p < 0.005 versus Rev-MEKE; comparisons among PDGFBB-stimulated cells: **, p < 0.0001 versus Rev-MEKE; n = 3).
27
Fig. 1: Arterial cell proliferation and atherogenesis in the arotic arch and femoral artery
of hypercholesterolemic rabbits. Rabbits received either control chow (n = 5) or a high-fat
diet (n = 10) for 2 months. Prior to sacrifice, animals were injected with BrdU to assess arterial
cell proliferation. The photomicrographs show representative examples of BrdU
immunoreactivity in cross-sections of the aortic arch (A) and femoral arteries (B) of fat-fed
rabbits. Specimens were counterstained with eosin. Two different magnifications are shown for
each specimen as indicated in each photomicrograph. Arrows in the 200x photomicrograph of
the femoral artery indicate two BrdU-positive cells within the intimal lesion. White arrowheads
point to the internal elastic lamina.
28
C
F
S
M
C
B
D
14000
*
6000
FSMC
2
4
6 hrs
***
FBS
30000
I
ASMC
***
20000
**
10000
***
120000
2
4
6 hrs
A
S
M
C
***
ASMC
†
80000
40000
FSMC
**
†
*
8
12
18
24
PDGF-BB
stimulus (hrs)
15
ASMC
10
FSMC
5
n = 267
FSMC
0
Hoechst 33258
F
S
M
C
0
0
Relative migration
H
ASMC
2000
0
Desmin
F
PDGF-BB *** ***
10000
E
TUNEL positive cells (%)
G
SM -actin
3
H-Thymidine
incorporation (cpm)
A
Relative migration
A
S
M
C
187
control
311
255
UV-irradiated
Fig. 2: Phenotypic differences between ASMCs and FSMCs. Phase contrast microscopic
view of primary cultures of ASMCs (A) and FSMCs (B) (200x magnification). (C-F) Indirect
immunofluorescence analysis of passage 2 primary cultures of ASMCs (C,E) and FSMCs (D,F)
(400x magnification). ASMCs and FSMCs disclosed similar expression of SM-actin (C,D).
By contrast desmin expression was low in ASMCs (E) and high in FSMCs (F). The right panel
in E and F shows nuclear staining (Hoechst 33258) in the same field shown for desmin staining
(left). Phenotypic differences between ASMCs and FSMCs were maintained up to passage 8.
(G) Primary cultures (passage 6) were labeled with the fluorescent dye Calcein AM and were
placed in serum-free media in the upper chamber of FALCON HTS FluoroBlock inserts. The
lower chamber contained either serum-free media, 10 ng/mL PDGF-BB (upper panel) or 10%
FBS (lower panel). Chemotaxis was assessed by detecting the fluorescence of cells migrating to
the lower chamber at the indicated time points after plating the cells. Results represent the
average fluorescence of PDGF-BB-induced or 10% FBS stimulated cells after subtracting the
fluorescence of unstimulated cells (n = 3). Differences were evaluated using ANOVA and
Fisher’s post hoc test. Only comparisons versus t = 0 are shown. * p < 0.05, **, p < 0.005, and
***, p < 0.0001. (H) Cells were maintained for 72 h in mitogen-free ITC media and then were
exposed to 10 ng/mL PDGF-BB for the indicated time. Cultures were pulsed with 3Hthymidine. Results represent the average of 3 experiments using passage 3, 4 and 6 cultures.
Differences were evaluated using ANOVA and Fisher’s post hoc test. Comparisons versus t =
0: *, p < 0.025, **, p < 0.015, ***, p < 0.0001; comparisons between ASMC and FSMC at each
time point: †, p < 0.0001 (n = 6 each time point). (I) Percentage of TUNEL-positive cells in
starvation-synchronized cultures. Analysis included control and UV-irradiated cells. The total
number of cells analyzed in 10 high-power fields (400x) is indicated below each bar.
29
Relative migration
A
15000
*
*
*
*
ASMC
*
9000
*
3000
0
0
2
B
3H-Thymidine
incorporation (cpm)
*
ASMC
12000
CSMC
*
4
6 hrs
*
†
*
CSMC
8000
4000
0
24
18
PDGF-BB stimulus (hrs)
Fig. 3: ASMCs and CSMCs display similar migratory and proliferative capacity.
Statistical analysis was performed using ANOVA and Fisher’s post hoc test. (A) Migration was
assayed as described in Fig. 2G using 10% FBS as the chemotactic agent. *, p < 0.0001 (versus
t = 0). (B) 3H-thymidine incorporation was assayed as indicated in Fig. 2H (n = 4 each time
point). *, p < 0.0001 (versus t = 0); †, p < 0.001 (comparisons between ASMC and CSMC at
each time point).
30
A
B
kDa
35
PDGF-BB stimulus (hrs)
0
3
9
0
3
2.1 1.8
ASMC
1
0
3
9
18
0
3
9
18
PDGF-BB
stimulus (hrs)
CDK2
9
nd nd 0.11 0.26 nd nd nd 0.19
Relative
activity
0.6 0.7
cyclin D1
35
32P-Histone
1
FSMC
ASMC
cyclin A
52
FSMC
Relative cyclin D1
nd nd 0.14 0.16 nd nd nd 0.07
Relative cyclin A
81
Ponceau
staining
52
C
kDa
0
3
6
35
FSMC
ASMC
9
0
3
6
9
PDGF-BB
stimulus (hrs)
112
D
kDa
PDGFR-
35
49
p27 (total)
CDK2
35
28
-tubulin
35
28
Phospho-p27
(Thr187)
50
-tubulin
E
p27
28
1
1 1.8 nd 6.5 3.7 5.5 1.6
C C
SM FSM
A
Relative p27
35
28
WB
IP: anti-Ubiquitin
WB: anti-p27 (total)
Fig. 4: ASMCs and FSMCs display dissimilar cell cycle regulatory protein expression and
CDK activation. Confluent cultures were maintained for 72 h in mitogen-free ITC media and
then exposed to 10 ng/mL PDGF-BB as indicated. Cell extracts were prepared in lysis buffer A
containing phosphatase inhibitors (A,B,D,E) or buffer B (C), which did not contain
phosphatase inhibitors. The analysis of lysates included cyclin A/cyclin E-associated CDK
activity using histone H1 and -32P-ATP substrates (A), Western blot with the indicated
antibodies (B,C,D), and immunoprecipitation with an anti-ubiquitin antibody followed by
Western blot of the immunoprecipitated material using anti-p27 antibodies (E). (A) Kinase
reactions were analyzed by SDS-PAGE and autoradiography. Relative activity was estimated
after densitometric analysis (0 hrs is set as 1 for each cell type). (B) Densitometric analysis was
performed to estimate the relative level of cyclin D1 and A. Each cyclin value was divided by
its corresponding CDK2 loading control. Shown below is the PVDF membrane stained with
Ponceau prior to incubation with antibodies(C) Densitometric analysis was performed to
estimate the relative p27 level. Each p27 value was divided by its corresponding tubulin
loading control (ASMC at 0 hrs = 1; nd: not detected). (D, E) The phospho-specific anti-p27
antibody only recognizes p27 phosphorylated on Thr187. Open and closed arrowheads point to
the slow and faster migrating p27 immunoreactive band, respectively. Note that the slow
migrating band that undergoes phosphorylation on Thr187 and ubiquitinilation prevailed in
ASMCs. By contrast, the faster migrating p27 band that does not contain protein
phosphorylated on Thr187 and does not undergo ubiquitination predominated in FSMCs (see
text for details).
31
B
6000
Relative migration
9000
*
kDa
Rev-p27
3000
Rev-LacZ
incorporation (cpm)
3H-thymidine
A
8000
**
6000
Rev-LacZ
*
4000
2000
†
††
0
Rev-p27
-2000
0
2
4
6 hrs
35
p27
28
-tubulin
50
1
3
Relative p27
Fig. 5: Inhibition of ASMC proliferation and migration by retrovirus-mediated p27
overexpression. ASMCs were infected with control retrovirus (Rev-lacZ) or a retrovirus
encoding for p27 (Rev-p27). Infected cells were selected with puromycin. (A) Cells were
maintained in 10% FBS/DMEM, pulsed for 4 h with 3H-thymidine and radioactivity
incorporated into DNA was quantified. Differences were evaluated using two-tail, unpaired
Student’s t test (*, p < 0.0015, n = 5). Puromycin-resistant cells were also lysed in buffer A to
perform Western blot analysis using anti-p27 and anti--tubulin antibodies. Densitometric
analysis was performed to estimate the relative level of p27. Each p27 value was divided by its
corresponding tubulin loading control (Rev-LacZ = 1). (B) Migration of ASMCs infected with
Rev-LacZ or Rev-p27 was measured as indicated in Fig. 2G using 10% FBS as the chemotactic
agent. Differences were evaluated using ANOVA and Fisher’s post hoc test (comparisons
versus t = 0: *, p < 0.005, **, p < 0.0001; comparisons between Rev-LacZ and Rev-p27 at each
time point: †, p < 0.02, ††, p < 0.0001).
32
ASMC
0
5
FSMC
15 30
0
5
15
30
P-ERK1
P-ERK2
PDGF-BB
stimulus
(min)
ERK1
ERK2
1
2.3 1.7 1.7 0.2 1.4 1.1 0.3
P-ERK / ERK
Fig. 6: ASMCs and FSMCs display dissimilar MAPK regulation. Western blot
analysis of cells maintained for 72 h in mitogen-free ITC media and then exposed to
10ng/mL PDGF-BB for the indicated time. Cell lysates were prepared in lysis buffer A.
P-ERK1/2 and ERK1/2 indicate phosphorylated (active) and total ERK1/2, respectively.
Densitometric analysis was performed to estimate the relative level of P-ERK1/2. Each
P-ERK value was divided by its corresponding ERK loading control (ASMC at 0 hrs =
1).
33
0 15 30 60 90 0 15 30 60 90
P-ERK1
P-ERK2
PDGF-BB
stimulus
(min)
kDa
nt
r
P D ol
98
05
9
B
PD98059
Co
Control
A
35
29
ERK1
ERK2
p27
 -tubulin
50
nd 1.7 1.2 0.7 0.6 0.2 1.1 0.6 nd 0.1
C
P-ERK / ERK
D
1
4.3
Mitogen-depleted
Relative p27
PDGF-BB
incorporation
(cpm)
40000
*
3H-thymidine
3H-thymidine
incorporation
(cpm)
PDGF-BB + PD98059
*
20000
†
*
††
0
*
*
15000
†
5000
0
2
10
50
PD98059 )
†
12
18
PDGF-BB stimulus (hrs)
Fig. 7: MAPK inhibition upregulates p27 expression and inhibits PDGF-BB-dependent
ASMC proliferation. Differences were evaluated using ANOVA and Fisher’s post hoc test.
(A, B) ASMCs were maintained for 72 h in ITC media and then were exposed to 10 ng/mL
PDGF-BB for short (A) or long periods of time (8 hrs, B). Cell lysates were prepared in lysis
buffer A to perform immunoblot analysis with the indicated antibodies. Treatment with 50 M
PD98059 was initiated 1 h before addition of PDGF-BB. P-ERK1/2 and ERK1/2 indicate
phosphorylated (active) and total ERK1/2, respectively. Densitometric analysis was performed
to estimate the relative level of P-ERK1/2 and p27. Each P-ERK or p27 value was divided by
its corresponding loading control (total ERK or tubulin, respectively; nd: not detected). For
p27, results are shown relative to control (set as 1). (C) Asynchronously growing ASMCs were
treated for 1 h in mitogen-free ITC media supplemented with PD98059 or vehicle, and then
cells were incubated for 24 h with 10 ng/mL PDGF-BB. Cultures were pulsed with 3Hthymidine during the last 4 h (n = 4). Comparisons versus control: *, p < 0.0001; comparisons
versus 2 M PD98059: †, p < 0.04, ††, p < 0.02. (D) Cells were maintained for 72 h in ITC
media and then were exposed to 10 ng/mL PDGF-BB. When indicated, mitogen-depleted
ASMCs were pretreated with 50 M PD98059 for 1 h prior to PDGF-BB stimulation. Cells
were pulsed with 3H-thymidine (n = 4,: *, p < 0.0001 versus control starvation-synchronized
cells; †, p < 0.0001 versus 50 M PD98059).
34
A
Relative migration
ASMC
**
*
14000
PDGF-BB
†††
6000
†
††
PDGF-BB
+ PD98059
0
0
B
3
5
FSMC
Relative migration
*
7 hrs
***
60000
Rev-MEKE
40000
**
*
20000
†
††
Rev-LacZ
0
4
6
8 hrs
Fig. 8: Altered MAPK signaling affects ASMC and FSMC migration. Migration assays
were performed as indicated in Fig. 2G using 10 ng/mL PDGF-BB (A) or 10% FBS (B) as the
chemotactic agent. Statistical analysis was done using ANOVA and Fisher’s post hoc test. (A)
ASMCs were untreated or exposed to 50 M PD98059 during labeling with calcein-AM.
PD98059 treatment was maintained in both the upper and lower chamber. Comparisons versus t
= 0: *, p < 0.05, **, p < 0.01; comparisons between control and PD98059 at each time point: †,
p < 0.05, ††, p < 0.02, †††, p < 0.006. (B) Migration of FSMCs infected with Rev-LacZ or
Rev-MEKE. Comparisons versus t = 0: *, p < 0.02, **, p < 0.002, ***, p < 0.0001;
comparisons between Rev-MEKE and Rev-LacZ at each time point: †, p < 0.04, ††, p <
0.0001.
35
C
Rev-MEKE
0 15 30 60 0
PDGF-BB
stimulus
(min)
15 30 60
P-ERK1
P-ERK2
ERK1
ERK2
nd nd
B
1
Rev-LacZ
kDa
0
3
6
1
0.9
2
Rev-MEKE
9
0
3
P-ERK / ERK
2.1 1.5
6
9
29
PDGF-BB
stimulus (hrs)
p27
 -tubulin
50
1
1.4 2.3 2.3 nd 0.2 nd nd
Uninfected
Rev-LacZ
incorporation (cpm x 10-3)
Rev-LacZ
3H-thymidine
A
Rev-MEKE
400
200
* *
0
*
* *
*
24
PDGF-BB stimulus (hrs)
Relative p27
Fig. 9: Forced MAPK activation downregulates p27 expression and stimulates PDGF-BBdependent FSMC proliferation. FSMCs were infected with control retrovirus (Rev-lacZ) or a
retrovirus encoding a constitutively active MEK1 mutant (Rev-MEKE). Infected cells were
selected with puromycin. Puromycin-resistant cells were maintained for 72 h in mitogen-free
ITC media, then cultures were stimulated with 10 ng/mL PDGF-BB for the indicated time. (A,
B) Cells were lysed in buffer A to perform immunoblot analysis. P-ERK1/2 and ERK1/2
indicate phosphorylated (active) and total ERK1/2, respectively. Densitometric analysis was
performed to estimate the relative level of P-ERK1/2 and p27. Each P-ERK and p27 value was
divided by its corresponding loading control (total ERK and tubulin, respectively; nd: not
detected). For p27, results are shown relative to Rev-LacZ at 0 hrs (set as 1). (C) 3H-thymidine
incorporation in mitogen-depleted cells (0 h) and 24 h upon PDGF-BB stimulation. Differences
were evaluated using ANOVA and Fisher’s post hoc test (comparisons among mitogendepleted cells: *, p < 0.005 versus Rev-MEKE; comparisons among PDGF-BB-stimulated
cells: **, p < 0.0001 versus Rev-MEKE; n = 3).
36