Báo cáo khoa học: Endogenous mono-ADP-ribosylation mediates smooth muscle cell proliferation and migration via protein kinase N-dependent induction of c-fos expression potx

Endogenous mono-ADP-ribosylation mediates smooth muscle cell
proliferation and migration via protein kinase N-dependent induction
of c-
fos
expression
Lorraine Yau
1,2
, Brenda Litchie
1
, Shawn Thomas
1
, Benjamin Storie
1
, Natalia Yurkova
1
and Peter Zahradka
1,2
1
Institute of Cardiovascular Sciences, St. Boniface Research Centre and
2
Department of Physiology, University of Manitoba,
Winnipeg, MB, Canada
ADP-ribosylation has been coupled to intracellular events
associated with smooth muscle cell vasoreactivity, cytoskel-
etal integrity and free radical damage. Additionally, there is
evidence that ADP-ribosylation is required for smooth
muscle cell proliferation. Our investigation employed
selective inhibitors to establish that mono-ADP-ribosylation
and not poly(ADP-ribosyl)ation was necessary for the sti-
mulation of DNA synthesis by mitogens. Mitogen treatment
increased concomitantly the activity of both soluble and

particulate mono-ADP-ribosyltransferase, as well as the
number of modified proteins. Inclusion of meta-iodo-
benzylguanidine (MIBG), a selective decoy substrate of
arginine-dependent mono-ADP-ribosylation, prevented the
modification of these proteins. MIBG also blocked the
stimulation of DNA and RNA synthesis, prevented smooth
muscle cell migration and suppressed the induction of c-fos
and c-myc gene expression. An examination of relevant
signal transduction pathways showed that MIBG did not
interfere with MAP kinase and phosphatidylinositol 3-kin-
ase stimulation; however, it did inhibit phosphorylation of
the Rho effector, PRK1/2. This novel observation sug-
gests that mono-ADP-ribosylation participates in a Rho-
dependent signalling pathway that is required for immediate
early gene expression.
Keywords: ADP-ribosylation; smooth muscle; DNA syn-
thesis; c-fos; MAP kinase.
Post-translational modification by ADP-ribosylation, the
enzymatic transfer of ADP-ribose from NAD
+
to an
acceptor protein, has been grouped into two distinct
classes that are distinguished by their reaction mechanisms
[1]. O-linked ADP-ribosylation of glutamate residues is
catalyzed by poly(ADP-ribose) polymerase (PARP-1), as
is the subsequent formation of polymers containing
10–100 ADP-ribose units. This process occurs primarily
in the nucleus, and is responsible for modulating DNA–
protein interactions [2]. It has been established that
poly(ADP-ribosyl)ation participates in DNA-base excision

repair [3], while PARP-1 degradation is a marker for
apoptosis [4]. PARP-1 has also been linked to differen-
tiation of neutrophilic cells [5], and may be important for
chromatin condensation [6], centromere function [7] and
transcription [8,9].
Unlike poly(ADP-ribosyl)ation, mono-ADP-ribosyla-
tion reactions involve the transfer of a single ADP-ribose
to various amino acid (arginine, histidine, diphthamide,
cysteine, asparagine) residues by mono-ADP-ribosyltrans-
ferases (mART) [10]. Investigations of bacterial toxins
(e.g. clostridia, cholera, pertussis, diphtheria) that exhi-
bited mART activity foreshadowed the identification of
endogenous enzymes capable of catalyzing similar reac-
tions [1]. The majority of vertebrate mARTs studied to
date have been found to modify either cysteine or
arginine. The cellular location of these enzymes has been
shown to vary, with enzymes detected in the cytosol,
microsomal and nuclear fractions [11]. Many membrane-
bound mARTs belong to the family of glycosylphospha-
tidylinositol (GPI)-anchored proteins present on the
extracellular surface. It has been proposed that the GPI-
anchored mARTs modulate transmembrane signalling
events, as integrin a7 is one target molecule that has
been identified [11]. In contrast, soluble ARTs have been
shown to modify cytoskeletal proteins such as actin, thus
interfering with polymerization [12]. Other critical medi-
ators of cellular function that undergo ADP-ribosylation
Correspondence to P. Zahradka, Institute of Cardiovascular Sciences,
Boniface Research Centre, 351 Tache Avenue, Winnipeg, MB,
Canada., Fax: +1 204 233 6723, Tel.: +1 204 235 3507,

E-mail:
Abbreviations: 3AB, 3-aminobenzamide; AngII, angiotensin II;
DMEM, Dulbecco’s modified Eagle’s medium; GPI, glycosylphos-
phatidylinositol; MAP kinase, mitogen-activated protein kinase;
MIBA, meta-iodobenzylamine; MIBG, meta-iodobenzylguanidine;
PD128763, 3,4-dihydro-5-methylisoquinoline; PGE
2
, prostaglandin
E
2
; PI3-kinase, phosphatidylinositol 3-kinase; PtdInsP
3
, phos-
phatidylinositol-3,4,5-trisphosphate; PRK, protein kinase C-related
kinase; PVDF, poly(vinylidene difluoride); SMC, smooth muscle cell.
Enzymes: poly(ADP-ribose) polymerase (EC 2.4.2.30); mono-ADP-
ribosyltransferase, arginine-dependent (EC 2.4.2.31); protein kinase
N/PRK1 (EC 2.7.1.37); extracellular signal-regulated (MAP) kinase
(EC 2.7.1.37); 1-phosphatidylinositol 3-kinase (EC 2.7.1.137);
Rho (EC 3.6.1.47).
(Received 21 August 2002, revised 21 October 2002,
accepted 14 November 2002)
Eur. J. Biochem. 270, 101–110 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03366.x
include various GTP-binding proteins, including G
s
,G
i
and Rho [13–15]. Although these target proteins suggest a
role for mono-ADP-ribosylation in signal transduction,
the functional significance of these modifications in

normal cell physiology, including cell proliferation and
differentiation, remains poorly understood.
Vascular smooth muscle cells (SMC) are required to
proliferate under pathological conditions and following
revascularization procedures that damage the vessel wall
[16]. Furthermore, this change in proliferation status is
preceded by a switch in cell phenotype [17]. Phenotypic
modulation, or the conversion of SMC to the less mature
phenotype, is reversible once the injury has been repaired
[18,19]. However, failure of SMC to revert to the mature
phenotype is associated with conditions such as athero-
sclerosis, hypertension and restenosis. For this reason,
interventions to treat these conditions have focused primar-
ily on the ability to interfere with either SMC migration or
proliferation.
Thyberg et al. [20] and Grainger et al. [21] have
reported that inhibitors of ADP-ribosylation prevent
phenotypic modulation, and consequently proliferation
of SMC. Furthermore, Thyberg et al. [20] concluded that
poly(ADP-ribosyl)ation and mono-ADP-ribosylation were
essential for the SMC response to mitogenic stimulation
based on evidence that inhibitors of both PARP-1 and
arginine-dependent mART prevented PDGF-stimulated
thymidine incorporation. Although a number of cell
processes were investigated, no definitive mechanism of
action was identified in these studies. We therefore
investigated the role of ADP-ribosylation in the SMC
response to mitogen stimulation in greater detail. Over
the course of this study, we established that activation of
an arginine-dependent mART, but not PARP-1, was

required for SMC proliferation, and identified a novel
link between mono-ADP-ribosylation and c-fos gene
expression.
Materials and methods
Materials
Cell culture materials (DMEM, fetal bovine serum,
trypsin, culture dishes) were obtained from Gibco/BRL,
as were oligonucleotide primers used for RT-PCR ampli-
fication. Poly(vinylidene difluoride) (PVDF) membrane
and DNA molecular mass markers were supplied by
Roche. The Boyden chamber and Track-Etch Membrane
polycarbonate filters used for cell migration assays were
purchased from Neuroprobe and Nucleopore, respectively.
The GeneAmp RT-PCR kit and radiolabelled chemicals
([
3
H]uridine, [
3
H]thymidine, [
3
H]NAD
+
,[
14
C]adenosine,
[
32
P]orthophosphate; manufactured by New England
Nuclear) were from Perkin Elmer-Cetus. SYBR Green I
was provided by Molecular Probes, while the BCA

Protein assay kit was supplied by Pierce-Endogen.
PD128763 was a generous gift from Parke-Davis. Other
chemicals, including mitogens and ADP-ribosylation in-
hibitors, were purchased from Sigma-Aldrich. Silica G
thin layer chromatography plates were from Whatman.
Phospho-specific antibodies (Elk1, PRK1/2) were obtained
from Cell Signaling Technology.
Smooth muscle cell culture
Primary cultures of porcine coronary artery SMC were
generated from the left anterior descending coronary artery
by an explant organ culture method [22] and propogated in
DMEM containing 20% fetal bovine serum. Cells were
used only after the second passage to maintain consistency
between cultures. Quiescence was achieved by incubating in
serum-free DMEM supplemented with 11 lgÆmL
)1
pyru-
vate, 5 lgÆmL
)1
transferrin, 1 n
M
selenium, 0.2 m
M
ascor-
bate and 10 n
M
insulin for 5 days.
DNA and RNA synthesis
Quiescent cells were prepared in 24-well dishes and stimu-
lated by direct addition of the indicated compounds without

replacing the media. When inhibitors were used, they were
added 10–15 min prior to the stimulating agents. To
measure RNA synthesis, cells were incubated for 6 h with
2 lCi [
3
H]uridine, added concomitantly with the stimulating
agent. Similarly, DNA synthesis was measured by incuba-
ting the cells with 2 lCi [
3
H]thymidine, added 24 h after
mitogen stimulation, for 48 h. Incorporation of radiola-
belled precursors into trichloroacetic acid-insoluble nucleic
acids was measured as described previously [23].
Western blotting
Extracts prepared by addition of 150 lL2· SDS/gel
loading buffer to cells in 12-well culture dishes were loaded
onto 7.5% polyacrylamide gels and the proteins were
subsequently transferred to PVDF membrane. The mem-
brane was probed with antibodies as described previously
[24]. Ponceau S staining was used to ensure equal protein
loading.
Cell migration
SMC were collected by trypsinization and 1 500 cells were
added to each well of a Boyden chamber (48-well unit)
containing a polycarbonate filter with 5 lm pores. Serum-
free DMEM with chemoattractant [1 l
M
angiotensin II
(AngII)] was added to the lower compartment while
inhibitors were added to the upper compartment. After

48hinastandardCO
2
incubator at 37 °C, the membrane
was removed from the chamber and placed into methanol
for 5 min Cells were scraped from the upper side of the
membrane and the membrane was subsequently placed into
Giemsa stain for 60 min The membrane was mounted on a
slide and the cells present on the underside of the membrane
were counted (n ¼ 6 per treatment).
Subcellular fractionation
SMC prepared and treated in 150-mm diameter tissue
culture dishes (Nunc) were washed twice in NaCl/P
i
(0.225
M
NaCl, 0.1
M
NaP
i
, pH 7.1), harvested by scraping in 3.0 mL
of NaCl/P
i
and collected by centifugation (5 min, 3 000 g at
4 °C). The cells were disrupted in 2.5 cell pellet volumes of
homogenization buffer (0.25
M
sucrose, 5 m
M
Tris/HCl,
pH 8.0, 3 m

M
CaCl
2
,1 m
M
EDTA, 0.5 m
M
EGTA, 0.2 m
M
phenylmethanesulfonyl fluoride, 25 kUÆmL
)1
aprotinin,
102 L. Yau et al. (Eur. J. Biochem. 270) Ó FEBS 2003
25% glycerol) using a Pro200 homogenizer (Pro Scientific
Inc.) fitted with a 5-mm generator. Nuclei were removed by
low-speed centrifugation (10 min at 8 000 g at 4 °C) and the
supernatant subsequently centrifuged (70 000 g for 60 min
at 4 °C) to separate the membrane and cytoplasmic
fractions. The microsomal pellet was resuspended with
200 lL homogenization buffer. Protein content was deter-
mined with the BCA protein assay kit was modified for a
96-well format to measure a 10-lL sample volume.
MAP kinase activity gel assay
Samples prepared from treated SMC by detergent lysis
[50 m
M
a-glycerphosphate pH 7.4, 0.5% (v/v) Triton
X-100, 25% (v/v) glycerol, 2 m
M
EGTA, 1 m

M
orthovana-
date, 1 m
M
dithiothreitol, 0.5 m
M
phenylmethanesulfonyl
fluoride, 0.1 m
M
bacitracin and 20 lgÆmL
)1
aprotinin] were
loaded (without heating) onto a 10% polyacrylamide gel
containing 0.5 mgÆmL
)1
myelin basic protein. Following
electrophoresis, phosphorylation of myelin basic protein
was assayed by incubating the gel with 25 lCi [c-
32
P]ATP in
50 m
M
Tris/HCl, pH 8.0, 2 m
M
dithiothreitol, 0.1 m
M
EGTA, 5 m
M
MgCl
2

and 100 l
M
ATP for 1 h at room
temperature [25]. The gel was then washed 5 · in 1%
sodium pyrophosphate/5% trichloroacetic acid, dried and
exposed to reflection film (Dupont) at )80 °C with one
intensifying screen.
Phosphatidylinositol 3-kinase assay
Quiescent cells were incubated with 200 lCiÆmL
)1
[
32
P]
orthophosphate for 4 h in phosphate-free media after a 1-h
preincubation in phosphate-free serum-free DMEM, as
described previously [26]. Cells were then stimulated for
15 min, with inhibitors added 10 min prior to addition of
agonist. Phosphatidylinositides were extracted after 5%
perchloric acid treatment and analyzed by thin layer chro-
matography on silica G plates developed in chloroform/
acetone/methanol/acetic acid/water (80 : 30 : 26 : 24 : 14,
v/v), and visualized by autoradiography.
Arginine-dependent mono-ADP-ribosyltransferase assay
Arginine-dependent mART was assayed by combining
70 lL assay buffer (70 m
M
Tris/HCl pH 7.5, 100 l
M
[
3

H]NAD
+
(0.25 lCi), 0.1 m
M
phenylmethanesulfonyl
fluoride), 10 lL polyarginine (2 mgÆmL
)1
)and20lL cell
extract (microsomal or cytosolic fraction) and incubating
this mixture for 30 min at 30 °C [27]. The reaction was
stopped by the addition of 1 mL cold 20% trichloroacetic
acid and the precipitate was captured on GF/C glass fibre
filters. The filters were washed extensively with 5% trichlo-
roacetic acid and the radioactivity was quantified by liquid
scintillation counting.
Metabolic labeling of mono-ADP-ribosylated proteins
Cells were incubated in 24-well dishes with 40 lCi
[
14
C]adenosine for 16 h in the presence or absence of
inhibitor [24]. The cells were washed once with ice-cold
NaCl/P
i
, and solubilized directly with 100 lL2· SDS/
gel loading buffer. The sample was then clarified by
centrifugation (12 000 g, 10 min) and an aliquot of 25 lL
was loaded onto a 10% polyacrylamide gel. The radio-
activity present in the gels was detected with a Molecular
Devices Storm phosphorimager.
Reverse transcription-polymerase chain reaction

amplification
Total RNA (1 lg) was amplified according to the protocol
recommended for the GeneAmp kit with oligodeoxynucle-
otide primers specific for GAPDH (sense: 5¢-CGGTGTG
AACGGATTTGGCCGTAT-3¢,antisense:5¢-AGCCTTC
TCCATGGTGGTGAAGAC-3¢); c-fos (sense: 5¢-GAATA
AGATGGCTGCAGCCAAGTGC-3¢,antisense:5¢-AAG
GAAGACGTGTAAGCAGTGCAGC-3¢), and c-myc
(sense: 5¢-AAGTTGGACAGTGGCAGGGT-3¢,antisense:
5¢-TTGCTCCTCTGCTTGGACAG-3¢). Amplification
was conducted over 35 cycles using a three-step program
as described previously [28]. Samples were analyzed by
electrophoresis in 1.7% agarose gels and visualized with
SYBR Green I.
Data measurement and statistical analysis
Radiotracer, cell number and enzyme assay data were
quantified and plotted as means ± SEM of individual
experiments (n ¼ 3–6). Student’s t-test was used to compare
treatment means vs. controls. Statistical significance was
set at P < 0.05. Quantification of data obtained on film
or autoradiographs was accomplished with a Bio-Rad
Model-670 Imaging Densitometer under nonsaturating
conditions.
Results
meta
-Iodobenzylguanidine inhibits SMC proliferation
The incorporation of [
3
H]uridine and [
3

H]thymidine was
used to quantify the relative rates of RNA and DNA
synthesis, respectively. Quiescent SMC were pretreated with
meta-iodobenzylguanidine (MIBG), a decoy substrate of
arginine-dependent mono-ADP-ribosylation that conse-
quently reduces modification of endogenous protein targets
[29], for 10 min prior to addition of various growth
stimulating agents. MIBG exhibited a concentration-
dependent inhibition of both RNA and DNA synthesis in
SMC stimulated with AngII (1 l
M
), with complete inhibi-
tion observed at 25 l
M
(Fig 1A and C). Similarly, MIBG
inhibited both RNA and DNA synthesis in fetal bovine
serum-stimulated (2% v/v) SMC (Fig 1B and D), although
a decrease to basal levels was not achieved. This is likely due
to the magnitude of the SMC growth response elicited by
the multiple growth stimulating agents present in fetal
bovine serum. However, when MIBG was tested with
prostaglandin E
2
(PGE
2
)-stimulated (1 l
M
)SMC,the
degree of inhibition (Fig. 1E) was comparable to that seen
with AngII. The inability of meta-iodobenzylamine

(MIBA), an inactive analogue of MIBG, to inhibit DNA
synthesis, even at a concentration of 50 l
M
(Fig. 1F),
confirmed the specificity of MIBG. Confirmation that cell
death is not the mechanism by which MIBG inhibits SMC
proliferation was obtained by examining the effect of MIBG
Ó FEBS 2003 ADP-ribosylation and SMC proliferation (Eur. J. Biochem. 270) 103
on cell morphology, which revealed no visible changes at
concentrations below 500 l
M
(Fig. 2).
Activation of arg-mART by mitogens
While the ability of MIBG to inhibit cell proliferation
suggests mono-ADP-ribosylation is required for cell cycle
progression, direct evidence to support this correlation is
limited. Furthermore, there is little experimental support to
show that mono-ADP-ribosylation can be activated in
response to mitogens. Therefore, arginine-dependent
mART activity was measured in extracts of SMC that
had been stimulated with either AngII or PGE
2
.Itwas
observed that both cytosolic and microsomal arg-mART
activity were increased transiently following AngII (1 l
M
)
stimulation (Fig. 3A), with cytosolic activity 1.78-fold over
basal levels at 30 min (Fig. 3B). Likewise, PGE
2

(1 l
M
)
treatment stimulated microsomal arg-mART (1.35-fold),
however, the increase in cytosolic activity was only marginal
(Fig. 3B).
Activation of arginine-dependent mART would be
expected to lead to an increase in the post-translational
modification of select proteins. Quiescent (5 days under
serum-free conditions) and growing cells (continuously in
fetal bovine serum) were therefore incubated for 16 h
with [
14
C]-adenosine. This approach was employed
because (a) cells are impermeable to NAD
+
,and(b)
Fig. 2. Effect of MIBG and MIBA on the Morphology of Quiescent
SMCs. Quiescent SMC were treated with MIBG or MIBA for 72 h.
Photomicrographs were used to record cell morphology. Representa-
tive micrographs are shown. Specific treatments are: untreated control
(A), 50 l
M
MIBA (B) and MIBG (C), 100 l
M
MIBA (D) and MIBG
(E), 200 l
M
MIBA (F) and MIBG (G), and 500 l
M

MIBA (H) and
MIBG (I). Magnification, 120 ·.
Fig. 1. Sensitivity of mitogen-stimulated SMC growth to MIBG. Qui-
escent SMC were pretreated with MIBG for 10 min prior to addition
of AngII (1 l
M
) (A and B), serum (2% v/v fetal bovine serum) (C and
D) or PGE
2
(1 l
M
) (E). The incorporation of [
3
H]uridine or
[
3
H]thymidine into trichloroacetate-precipitable material after addition
of AngII and fetal bovine serum was used to monitor RNA and DNA
synthesis, respectively. In panel F, [
3
H]thymidine incorporation by
quiescent SMC treated with PGE
2
(1 l
M
) was measured after pre-
treatment with MIBA rather than MIBG. The incorporation rate of
untreated cells was set to 100%. Bars show means + SEM (n ¼ 3) of
separate experiments conducted with three different SMC isolations.
Statistically significant differences relative to maximally stimulated

cells(minusMIBG)areindicated(*P <0.05).
104 L. Yau et al. (Eur. J. Biochem. 270) Ó FEBS 2003
permeabilization techniques stimulate poly(ADP-ribo-
syl)ation that masks the less abundant mono-ADP-
ribosylation. Numerous labeled proteins were detected in
quiescent SMC (Fig. 4), however, both the number of
bands and the labeling intensity were increased in
growing cells. The most prominent increases were in
bands of 140, 133, 116, 95, 82, 73 and 56 kDa (Fig. 4).
Other bands also showed differences between the
quiescent and growing populations, but these were not
as pronounced. Interestingly, a 60-kDa band detected in
quiescent cells was absent in the growing cell population.
Inclusion of 50 l
M
MIBG during the incubation
period with the growing cells caused a reduction in
labeling of the 140, 133, 116, 95, 82 and 73 kDa
proteins, while the labeling of several proteins
(66, 56 kDa) was unchanged. The insensitivity of the
56 and 66 kDa bands to MIBG can most likely be
attributed to modification of amino acids other than
arginine [10]. Alternatively, these proteins may have been
subjected to another form of post-translational modifi-
cation that leads to attachment of labeled adenosine
(e.g. adenylylation). Nevertheless, these data establish for
the first time that arginine-dependent mono-ADP-ribosy-
lation is responsive to mitogens, and that MIBG directly
inhibits this process.
Fig. 3. Activation of mono-ADP-ribosyltransferase by angiotensin II

and prostaglandin E
2
. Quiescent SMC were treated with AngII (1 l
M
)
or PGE
2
(1 l
M
) over 120 min and subcellular fractions prepared as
described in Materials and methods at the specified times. (A)
Microsomal (membrane) and cytosolic (cytosol) fractions of AngII-
stimulated SMC were assayed for arg-mART activity as described in
Materials and methods with polyarginine (2 mgÆmL
)1
) as the acceptor
molecule. (B) Mono-ADP-ribosylation was measured in vitro with
microsomal (membrane) and cytosolic (cytosol) fractions prepared
30minafterAngIIandPGE
2
(conditions equivalent to panel A)
treatment. Activity of the untreated cell extracts was set to 1.0. Bars
show means + SEM for three independent experiments. Statistically
significant differences relative to the respective unstimulated controls
(*P < 0.05) are indicated in both panels.
Fig. 4. Protein modification with mono-ADP-ribose. Quiescent (Q) and
growing (G) SMC were incubated with [
14
C]adenosine for 16 h as
describedinMaterialsandmethods.MIBG(50l

M
)wasincludedfor
the entire incubation period with a growing cell sample (M). Cellular
protein was extracted, separated by SDS/PAGE and tagged proteins
visualized as described in Materials and methods. Positions of the
molecular mass markers are indicated on the right side, while estimated
molecular masses of labeled proteins that have changed intensity in
relation to growth state are presented on the left. Data are represen-
tative of three independent experiments, each of which exhibited
similar results.
Ó FEBS 2003 ADP-ribosylation and SMC proliferation (Eur. J. Biochem. 270) 105
Participation of poly(ADP-ribosyl)ation
in cell proliferation
The incorporation of [
3
H]thymidine was used to compare
the effect of a selective inhibitor of poly(ADP-ribosyl)ation,
PD128763 [24], on the stimulation of DNA synthesis by
mitogen (2% fetal bovine serum). PD128763 was unable to
prevent the increase in thymidine incorporation obtained
following mitogen stimulation (Fig. 5). 3-Aminobenzamide
(3AB), a weak inhibitor of both PARP-1 and mART, was
also tested for comparative purposes [24] and was found to
reduce thymidine incorporation only slightly at a concen-
tration of 5 m
M
. In contrast, DNA synthesis was inhibited
completely in the presence of 50 l
M
MIBG (Fig. 5). These

data suggest that poly(ADP-ribosyl)ation is not required for
SMC proliferation.
Effect of MIBG on AngII-mediated SMC migration
As MIBG prevents the proliferation of SMC, it was also of
interest to determine if MIBG could inhibit SMC migration.
AngII (10 l
M
) is a potent chemoattractant when tested in a
Boyden chamber assay (Fig. 6). Inclusion of MIBG (50 l
M
)
significantly reduced SMC migration relative to AngII
alone. These data demonstrate that MIBG inhibits SMC
migration in addition to SMC growth. The ineffectiveness
of other inhibitors (3AB, PD128763) indicated that poly
(ADP-ribosyl)ation is not connected to SMC migration.
MIBG prevents induction of c-
fos
gene expression
by mitogens
Increased c-fos gene activity is a hallmark of mitogen
stimulation, and RT-PCR of total RNA was therefore used
to monitor the effect of MIBG on c-fos expression. As
expected, both PGE
2
(Fig. 7A) and AngII (data not shown)
transiently elevate c-fos mRNA levels within 15 min post-
stimulus. Pretreatment of SMC with MIBG, however,
prevented the increase in c-fos mRNA levels obtained in
response to PGE

2
in a concentration-dependent manner
(Fig. 7A). Quantitative analysis of these data confirmed
that 50 l
M
MIBG was sufficient to inhibit PGE
2
-dependent
c-fos gene expression by 82%. Similarly, 50 l
M
MIBG
prevented expression of c-myc at 90 min when measured by
RT-PCR (Fig. 7B). These observations indicate that a step
required for induction of c-fos gene transcription, and
consequently c-myc expression, is sensitive to MIBG
treatment and may be regulated by an arginine-dependent
mART.
Inhibition of intracellular signalling by MIBG
The induction of c-fos gene expression involves multiple
pathways, each activating an essential transcription factor
that binds to the c-fos promoter. MAP kinase, for instance,
is necessary for phosphorylation of Elk1. Therefore, to
determine whether the inhibition of growth effect of MIBG
is due to an effect on MAP kinase activation, MIBG
(50 l
M
) was added to SMC 10 min prior to growth factor
stimulation. Under these conditions, MIBG did not prevent
PGE
2

-stimulated phosphorylation of myelin basic protein
by MAP kinase (Fig. 7C). Rather, there was a slight
stimulation in the presence of MIBG for which there is no
explanation. Nevertheless, as this increase in MAP kinase
activity was observed in all experiments, it may be
speculated that MIBG inhibits a negative regulator of this
Fig. 5. Effect of ADP-ribosylation inhibitors on DNA synthesis. Qui-
escentSMCwerepreparedin24-welldishesandstimulatedwithPGE
2
(1 l
M
). ADP-ribosylation inhibitors (5 m
M
3-aminobenzamide, 10 l
M
PD128763, 50 l
M
MIBG) were added 15 min prior to mitogenic sti-
mulation. Thymidine incorporation was monitored as described in
Materials and methods. Bars show means + SEM (n ¼ 3). The results
were reproducible with three different SMC isolations. Statistically
significant differences relative to control (*P < 0.05) and PGE
2
-sti-
mulation (+P < 0.05) are indicated.
Fig. 6. Inhibition of SMC migration by ADP-ribosylation inhibitors.
Growing SMC were placed into a Boyden chamber and directional
movement of SMC through a membrane with 5 lmporestowards
AngII (1 l
M

) was monitored as described in Materials and methods.
SMC on the underside of the membrane were fixed, stained and
counted. Inhibitors (5 m
M
3-aminobenzamide, 10 l
M
PD128763,
50 l
M
MIBG) were in the upper chamber. Bars show means + SEM
(n ¼ 6). The histogram represents one of two independent experiments
using different SMC isolations. Statistically significant differences
relative to control (*P < 0.05) and to AngII-stimulated cells in the
absence of inhibitor (+P < 0.05) are indicated.
106 L. Yau et al. (Eur. J. Biochem. 270) Ó FEBS 2003
cascade. Like MAP kinase, PI3-kinase is also required for
c-fos expression [26], and is activated by both PGE
2
(1 l
M
)
and thromboxane A
2
(0.1 l
M
). However, pretreatment of
SMC with MIBG (50 l
M
) similarly did not inhibit PGE
2

-
dependent formation of PtdInsP
3
by PI3-kinase (Fig. 7D).
These results suggest pathways leading to Elk1 are not
sensitive to MIBG, and this was confirmed by showing
MIBG did not inhibit Elk1 phosphorylation in response to
mitogen stimulation (Fig. 7E). In contrast with MAP
kinase, Rho mediates growth factor-dependent activation
of c-fos via the serum response factor (SRF) [30]. Protein
kinase C-related kinase 1 (PRK1), also termed protein
kinase N (PKN), and PRK2 have been identified as critical
intermediates linking Rho to SRF [31]. Mitogen stimulation
of SMC resulted in phosphorylation of both PRK1 and
PRK2, which is indicative of their activation (Fig. 7F),
while MIBG inhibited this phosphorylation event. These
data suggest that the growth inhibitory actions of MIBG
result from its ability to prevent Rho-dependent activation
of SRF.
Discussion
The results of this study indicate that an arginine-dependent
mART is activated upon stimulation of SMC with growth
factors such as serum, AngII and PGE
2
,andthatMIBG,a
selective decoy substrate of arginine-dependent mARTs that
competes with the arginine moiety of target proteins [29],
prevents SMC proliferation and migration following treat-
ment with these growth factors. Furthermore, our novel
Fig. 7. Effect of MIBG on prostaglandin E

2
-stimulated gene expression and intracellular signalling pathways. (A) Quiescent SMC were treated for
15minwithPGE
2
(1 l
M
)subsequenttoa10-minpretreatmentwithMIBG(50l
M
). RNA was extracted and c-fos mRNA levels assessed by RT-
PCR as described in Materials and methods. Molecular mass markers (DNA Marker VI) were used to confirm the size of the PCR products. RNA
loading was confirmed by concurrent amplification of GAPDH. One representative agarose gel is shown. (B) Quiescent SMC were treated as
described in panel A, and total RNA was harvested at 90 min and RT-PCR amplification was used to determine c-myc expression. RNA loading
was confirmed by concurrent amplification of GAPDH. One representative agarose gel is shown. The results presented in panels A and B were
confirmed in two independent experiments using different SMC isolations. (C) MAP kinase activity was measured by activity gel assay over 20 min
after treatment with PGE
2
(1 l
M
). Cells were pretreated with MIBG (50 l
M
) for 10 min prior to PGE
2
stimulation. Specific phosphorylation of
myelin basic protein by p42
MAPK
(p42) and p44
MAPK
(p44) is shown. One of three independent experiments with different SMC isolations is
presented. (D) Phosphate pools in quiescent SMC were labelled with 200 lCi [
32

P]orthophosphate for 4 h prior to treatment with MIBG (50 l
M
)
for 10 min followed by addition of PGE
2
(1 l
M
)orTxA
2
(0.1 l
M
) for 15 min. Phosphoinositides were extracted from the cells and the phos-
phorylated forms of phosphoinositol were resolved by thin layer chromatography (TLC) as described in Materials and methods. A representative
autoradiogram of a TLC plate is shown, with PtdInsP
1
,PtdInsP
2
and PtdInsP
3
indicated. These results were confirmed in two independent
experiments. (E) Quiescent SMC were treated for 15 min with PGE
2
(1 l
M
)subsequenttoa10-minpretreatmentwithMIBG(50l
M
). Proteins
were extracted and analyzed by Western blotting. Detection of phosphorylated Elk1 is shown. One of three independent experiments with different
SMC isolations is presented. (F) Phosphorylation of PRK1/2 was measured by Western blotting as described for panel E. Three independent
experiments employing different SMC isolations were tested, and one result is presented in this panel.

Ó FEBS 2003 ADP-ribosylation and SMC proliferation (Eur. J. Biochem. 270) 107
findings suggest that mono-ADP-ribosylation is essential
for the induction of c-fos, and subsequently c-myc,gene
expression in response to certain mitogenic stimuli, possibly
via a Rho-dependent process. These observations therefore
implicate arginine-dependent mono-ADP-ribosylation in
the transduction of signals from cell surface receptors to the
nucleus.
In addition to establishing a role for mono-ADP-
ribosylation in SMC proliferation and migration, our data
also confirm that poly(ADP-ribosyl)ation does not partici-
pate in these events. While consistent with our study of
insulin-dependent proliferation of H4IIE hepatoma cells
[24], this conclusion does not concur with Thyberg et al.[20]
who found poly(ADP-ribosyl)ation is necessary for SMC
growth. Their reasoning was based on evidence that HMBA
(hexamethylenebisacetamide) blocked the transition of rat
aortic SMC from a contractile to a synthetic phenotype, in
addition to inhibiting DNA synthesis, protein synthesis and
the expression of several genes. Although inhibition of SMC
proliferation by HMBA has been reported in two inde-
pendent studies [20,21], the relationship between these
actions and the inhibition of PARP-1 remains tenuous. In
fact, numerous other intracellular targets for HMBA have
been proposed [32], including SMC-derived TGF-a [33]. As
well, the inability of 3AB to effectively inhibit cellular
proliferation [29,34], as we have shown for SMC (Fig. 5),
further supports the inference that poly(ADP-ribosyl)ation
does not participate in this process. Finally, the ineffective-
ness of PD128763 (Fig. 5), a specific PARP-1 inhibitor at

the concentrations employed in this study [24], shows
convincingly that poly(ADP-ribosyl)ation has no role in
either SMC proliferation or migration.
Uncoupling poly(ADP-ribosyl)ation from SMC prolifer-
ation was anticipated given the results of our previous study
[24]. Similarly, as MIBG can prevent the proliferation of
multiple cell types [35], its effect on SMC was also expected.
However, the mechanism by which MIBG inhibits cell
growth has yet to be resolved, although it has been
ostensibly attributed to an inhibition of either mitochondrial
respiration or mono-ADP-ribosylation [35,36]. In this
study, we have shown that MIBG blocks an early step
leading to expression of the c-fos and c-myc genes (Fig 7A
and B). This novel observation is clearly inconsistent with a
general effect on mitochondrial activity. Strong support for
this view is also provided by the fact that growth inhibition
is dissociated from cell death, as MIBG-induced changes in
SMC morphology are not evident at concentrations below
500 l
M
(Fig. 2).
The most significant outcome of this study was identify-
ing a link between MIBG and c-fos gene expression, and
therefore implies ADP-ribosylation regulates induction of
the c-fos gene. Although this finding contrasts with the
observation of Thyberg et al. [20] who reported that MIBG,
at a concentration of 100 l
M
, did not inhibit induction of
either c-fos or c-myc gene expression, this discrepancy may

be correlated with the limited potency of MIBG (e.g. DNA
synthesis was inhibited by only 50%) under their experi-
mental conditions. Furthermore, the distinct methods used
to prepare the cell cultures (collagenase digestion vs. explant
culture), as well as the time in reduced serum media (48 h vs.
5 days) intended to produce a quiescent state, may
contribute to differences in their response to mitogenic
stimuli. In any case, the data presented in Fig. 1 indicate the
concentration of MIBG required to inhibit SMC prolifer-
ation is in agreement with the earlier studies that first
reported MIBG inhibits cell proliferation [35,36].
Two potential routes by which MIBG could influence
c-fos gene expression were examined. Their selection was
based on information that each pathway is known to
activate one of the key transcription factors that regulates
c-fos promoter activity. MAP kinase and PI3-kinase were
investigated because it has been established they couple
immediate early gene induction to mitogenic signals gener-
ated by both AngII and PGE
2
[26]. MAP kinase operates
through Elk1, one component of the transcriptional
machinery required for c-fos gene activity [37]. The lack of
inhibition of MAP kinase activation (Fig. 7C) and Elk1
phosphorylation (Fig. 7E) by MIBG, however, suggests this
pathway is independent of ADP-ribosylation. Expression of
the c-fos gene also requires SRF, which activates c-fos gene
transcription via the serum response element located at
) 300 relative to the site of transcription initiation [38].
Inhibition of PRK1/2 phosphorylation by MIBG could

imply that SRF activation is ADP-ribosylation-dependent.
This connection is based on published evidence linking
PRK1/2 with SRF [31]. Furthermore, the fact that PRK1/2
is a downstream effector of Rho suggests ADP-ribosylation
may contribute to the regulation of Rho-dependent signal-
ling [39]. Accordingly, an association between Rho and
ADP-ribosylation has been established previously [15]. In
addition, inhibition of Rho by MIBG could explain the
diverse effects of this compound on both cell proliferation
and migration, as Rho is necessary for both processes [40].
Nevertheless, the exact target that mediates the presumed
mono-ADP-ribosyltransferase function, however, remains
to be identified. However, given the ability of ADP-
ribosylation to regulate GTP/GDP-binding status of
numerous proteins [41], it is possible to speculate that one
of the GTP-binding proteins responsible for Rho activation
(e.g. G
12/13
-dependent activation of p115 RhoGEF [42]), or
possibly Rho itself, is the most likely target.
It is also noteworthy that inhibition of Rho activation
influences cytoskeletal organization and thus cell motility.
Indeed, the GTP-binding proteins Rho, Rac and cdc42,
which regulate the cytoskeletal rearrangements necessary
for migration, are known targets of toxin-mediated mono-
ADP-ribosylation [41]. On the other hand, other possible
targets should also be considered, including actin, desmin
and tubulin, as mono-ADP-ribosylation has been shown
to influence their assembly/disassembly [43–46]. Further
investigation will be necessary to identify the mechanism by

which MIBG inhibits cell migration.
This investigation provides the first evidence to directly
link mono-ADP-ribosylation with SMC proliferation and
migration. Furthermore, expression of the c-fos gene was
the earliest proliferative event exhibiting sensitivity to
MIBG treatment, and represents a novel mechanism by
which mono-ADP-ribosylation can influence cellular
processes. As it is known that FOS mediates c-myc gene
activation, the inhibition of c-myc expression by MIBG
supports the premise that SRF-dependent c-fos gene
expression is the target of this inhibitor. Thus, c-fos
gene expression and PRK1/2 phosphorylation can now
be employed as endpoints for identification of those
108 L. Yau et al. (Eur. J. Biochem. 270) Ó FEBS 2003
cellular events that are susceptible to inhibition by MIBG
and should permit further examination of the role of
mono-ADP-ribosylation in cell cycle control and cell
proliferation.
Acknowledgements
This study was supported by grants from the Natural Sciences and
Engineering Research Council and by funding to the CIHR Group in
Experimental Cardiology. L. Y. was supported by a studentship from
the St Boniface Research Foundation.
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