(Received for publication, February 21, 1995; and in revised form, May 9, 1995)
From the
Angiotensin II (Ang-II) receptor engagement activates many
immediate early response genes in both vascular smooth muscle cells and
cardiomyocytes whether a hyperplastic or hypertrophic response is
taking place. Although the signaling pathways stimulated by Ang-II in
different cell lines have been widely characterized, the correlation
between the generation of different second messengers and specific
physiological responses remains relatively unexplored. In this study,
we report how in both C2C12 quiescent myoblasts and differentiated
myotubes Ang-II significantly stimulates AP1-driven transcription and
c-Junc-Fos heterodimer DNA binding activity. Using a set of
different protein kinase inhibitors, we could demonstrate that
Ang-II-induced increase in AP1 binding is not mediated by the
cAMP-dependent pathway and that both protein kinase C and tyrosine
kinases are involved. The observation that in quiescent myoblasts
Ang-II increase of AP1 binding and induction of DNA synthesis and, in
differentiated myotubes, Ang-II stimulation of protein synthesis are
abolished by the cysteine-derivative and glutathione precursor N-acetyl-L-cysteine strongly suggests a role for
reactive oxygen intermediates in the intracellular transduction of
Ang-II signals for immediate early gene induction, cell proliferation,
and hypertrophic responses.
Octapeptide Ang-II, ()a potent vasoconstrictor, is
also a growth factor for vascular smooth muscle cells
(VSMCs)(1, 2, 3, 4) . A number of in vivo and in vitro studies suggest that Ang-II may
also be a critical factor in mediating cardiac
hypertrophy(5, 6, 7, 8, 9) .
Hypertrophy is the fundamental adaptive process employed by postmitotic
cardiac and skeletal muscle in response to mechanical
load(10) . Using a load-induced cardiac hypertrophy in
vitro model, it has been recently demonstrated that mechanical
stretch causes the release of Ang-II from cardiac myocytes and that
locally produced Ang-II acts as the initial mediator of stretch-induced
hypertrophic response(11) .
In cardiac myocytes and
nonmyocytes, Ang-II induces immediate early genes such as
c-fos, c-jun, and egr1 leading to
hypertrophy and mitogenesis, respectively(12) . In general,
induction of immediate early genes is regulated by post-translational
modification of pre-existing factors and is directly regulated by
cellular second messenger systems(13) . Many peptide growth
factors, such as bombesin and endothelin-1, activate multiple second
messenger systems, which act synergistically to induce complex
mitogenic responses(14) . In different cell types a variety of
second messengers have been involved in the transduction of Ang-II
signaling. In cardiac myocytes and VSMCs Ang-II activates phospholipase
C through a G-protein-coupled receptor, liberates inositol
triphosphate; induces calcium release from inositol
triphosphate-sensitive calcium storage sites; activates protein kinase
C, phospholipase A, phospholipase D, adenilate cyclase, and
arachidonic acid metabolism; and stimulates the tyrosine kinases,
c-Raf1, and mitogen-activated protein kinases
cascade(12, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
Interestingly, the activation of both phospholipase A
and
phospholipase D stimulates the intracellular generation of ROIs through
the formation of arachidonate and phosphatidic acid (PA), respectively.
In turn, it has been suggested that they act as second messengers in
many physiological and pathological responses(24) , including
early response gene activation and cell growth
regulation(24, 25, 26) . However, the
interplay between all these transducers of Ang-II signaling and their
relation with specific responses is still unclear.
All muscle cell types share several structural properties and the expression of most of the known specific genes of muscles. These basic features are faithfully reproduced in primary cultures of fetal myoblasts and newborn satellite muscle cells, as well as in continuous mammalian myogenic cell lines. We used mouse C2C12 skeletal myoblasts, because they reproduce myogenic differentiation in culture(27) , to form long term differentiated and functional grafts in adult syngeneic ventricular myocardium (28) and to represent an attractive means of studying the effects of Ang-II in different conditions of proliferation and terminal differentiation. Systematic dissection of Ang-II transduction pathway in myogenic cells enabled us to show the involvement of ROIs in the intracellular transduction of Ang-II signals for immediate early genes induction, cell proliferation, and hypertrophic response.
The pBL2CATdel plasmid, containing the chloramphenicol acetyltransferase (CAT) gene driven by the minimal herpesvirus thymidine kinase (tk) gene promoter (positions -109 to +55), is described elsewhere(29) . The TRE-tk-CAT and the mtTRE-tk-CAT plasmids were derived from pBL2CATdel by inserting three copies of either a wild type or a mutated human collagenase TRE upstream from the tk promoter. To obtain the TRE-tk-CAT and the mtTRE-tk-CAT stable cell lines, C2C12 myoblasts were cotransfected by the calcium phosphate precipitation method with 5 µg of the CAT reporter plasmid and 250 ng of the neomycin resistance gene vector pAG60. After selection by G418 at 500 µg/ml for 14 days, individual clones were picked up and expanded. In unstimulated and Ang-II-stimulated cells, CAT activity was assayed as described(29) .
N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide, H7,
staurosporine, genistein, tyrphostin 25, and tyrphostin 1 were
dissolved in dimethyl sulfoxide and added to the cells 2 h before the
Ang-II stimulation to final concentrations of 75 mM, 100
mM, 70 ng/ml, 100 mM, 20 mM, and 20
mM, respectively. NAC and pyrrolidinedithiocarbamate were
dissolved in HO and added to the cells 1 h before the
Ang-II stimulation to final concentrations of 20 mM and 60
mM, respectively.
Figure 1:
Effects of Ang-II on
TRE-directed transcription in TRE-tk-CAT and mtTRE-tk-CAT stable C2C12
cell lines. Cells from both stable cell lines were plated at 2
10
/10-cm dish, cultured for 24 h, exposed for 24 h to
Ang-II, and lysed by four cycles of freezing and thawing. 50 µg of
total protein were assayed for CAT activity as described. The results
are expressed as fold activation (i.e. the ratio of the
percentage of conversion obtained in treated cells to the percentage of
conversion obtained in untreated cells). Experiments were repeated
three to five times with at least two different preparations of DNA. AII, Ang-II; `` + Sar,
[Sar
,Ile
]Ang-II.
Figure 2:
AP1 binding activity in C2C12 mouse
muscle cells. Cell extracts from actively growing or serum starved
``quiescent'' (A) and differentiated (B)
C2C12 cells were prepared at the indicated time points as described
elsewhere (28) . 5 µg of each extract were tested in gel
retardation assays with a P-5`-end-radiolabeled
oligonucleotide containing a canonical TRE site
(5`-TCGAGTGTCTGACTCATGCTTTCGA-3`). The specificity of the retarded
complexes was assessed by preincubating the extracts with increasing
amounts of cold specific TRE or unrelated NF
B probes. FCS, fetal calf serum.
Figure 3:
Induction by Ang-II of AP1 binding in
quiescent C2C12 myoblasts (A) and differentiated C2C12
myotubes (C). Cell extracts were prepared and tested as
described in gel retardation assays. The specificity of the retarded
complexes was assessed by preincubating the extracts with increasing
amounts of cold specific TRE or unrelated NFB probes. The relative
AP1 DNA binding activity (mean ± S.E. from three to five
experiments) was evaluated by laser densitometry analysis (GelScan,
Pharmacia Biotech Inc.) and expressed as fold activation with respect
to either unstimulated quiescent myoblasts (B) or unstimulated
differentiated myotubes (D). The composition of TRE-bound
complexes was evaluated by overnight preincubation of cell extracts
from Ang-II-treated quiescent myoblasts (B) or differentiated
myotubes (D) at 4 °C with 0.5 µg of a polyclonal
antibody against the epitope 128-152 of c-Fos (Santa Cruz
Biotechnology) and a polyclonal antibody directed against the
N-terminal domain of c-Jun (Oncogene Science Ab-2) not cross-reacting
with other Fos and Jun proteins. A cycloheximide concentration of 10
µg/ml was added to Ang-II-untreated and -treated cells (C and D). CHX, cycloheximide; FCS, fetal
calf serum; AII, Ang-II.
Ang-II strongly increases AP1
binding activity in both quiescent myoblasts (Fig. 3A)
and differentiated myotubes (Fig. 3C), and this
phenomenon is utterly inhibited by [Sar,
Ile
]Ang-II (Fig. 3, B and D).
Specific anti-c-Fos and anti-c-Jun antibodies were both able to almost
totally eliminate the TRE binding activity in Ang-II-treated C2C12
myoblasts and myotubes (Fig. 3, B and D),
suggesting that after Ang-II stimulation, most of TRE-bound AP1
proteins consist of c-Fos and c-Jun. A variety of control antibodies
(anti-p53, anti-E1A, anti-Myc) were unable to modify both basal and
induced patterns (Fig. 3, B and D, and data
not shown).
Figure 4:
Effects of protein kinase inhibitors and
anti-oxidants on Ang-II induction of TRE binding. Cell extracts were
prepared from unstimulated and Ang-II-stimulated C2C12 quiescent
myoblasts treated with different protein kinases inhibitors (A) and from HO
stimulated cells
treated with the cysteine-derivative and glutathione precursor NAC (B) (see ``Experimental Procedures''). Gel
retardation assays were performed, and the results are expressed as
described in Fig. 3. AII, Ang-II; HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; Tyr1,
tyrphostin 1; Tyr25, tyrphostin 25; Gen, genistein; FCS, fetal calf serum.
Figure 5:
Effects of NAC and H7 treatment on TRE DNA
binding induction by TPA (100 ng/ml) and HO
(200 µM) in C2C12 quiescent myoblasts. NAC and H7
have been used as described under ``Experimental
Procedures.'' The relative AP1 DNA binding activity (mean ±
S.E. from three to five experiments) was evaluated by laser
densitometry analysis (GelScan) and expressed as fold activation with
respect to either unstimulated quiescent myoblasts. FCS, fetal
calf serum.
Figure 6:
Ang-II induction of DNA synthesis in
quiescent C2C12 myoblasts. Cells were kept either in 0.1% serum for 36
h (myoblasts) or in 1% serum for 48 h (myotubes) and then stimulated
for 24 h with Ang-II at 10M or 200
uM H
O
in the presence or absence of
[Sar
,Ile
]Ang-II and NAC. Control
cultures were prepared without stimulation with Ang-II or
H
O
. In both preparations, for the last 18 h 10
uM bromodeoxyuridine was added. BrdU,
bromodeoxyuridine; FCS, fetal calf serum; AII,
Ang-II.
Figure 7:
Ang-II induction of protein synthesis in
differentiated C2C12 myotubes. Cells were kept either in 0.1% serum for
36 h (myoblasts) or in 1% serum for 48 h (myotubes), stimulated for 24
h with Ang-II at 10M or 200 uM
H
O
in the presence or absence of
[Sar
,Ile
]Ang-II and NAC, and finally
labeled with 200 mCi/ml [
S]methionine
(Trans-label). FCS, fetal calf serum; AII,
Ang-II.
Ang-II induces both proximal and distal signaling events
ultimately leading to cell growth in a variety of myogenic and
nonmyogenic cells. Ang-II binding to the Ang-II type 1 receptor
initiates a cascade of early biochemical cellular events similar to
those triggered by peptide growth factors. These include a rapid
production of diacylglycerol and inositol triphosphate by phospholipase
C-mediated hydrolysis of inositol phosholipids and activation of
protein kinase C(12, 17) , c-Raf1 serine threonine
kinase(21) , and mitogen-activating protein
kinases(12, 38) . Studies in rat liver epithelial
cells, renal mesangial cells, and VSMCs have demonstrated that Ang-II
stimulates tyrosine phosphorylation of several substrates, including
phospholipase C-1(19, 22, 38) . In
cardiac fibroblasts, it has been shown that Ang-II induces tyrosine
phosphorylation of the p125 focal adhesion kinase (p125
),
a dominant tyrosine kinase substrate, after stimulation of many
G-protein-coupled receptors and of p46
and
p56
. These play a crucial role in the activation of
p21
and serve as a converging target for both growth
factor and G-protein-coupled receptor-stimulated mitogenic
responses(23) . In VSMCs, Ang-II also causes a rapid and
sustained activation of phospholipase D-mediated phosphatidylcholine
hydrolysis, resulting in the formation of PA(39) . Although a
certain part of PA is converted to diacylglycerol, the PA that builds
up could be involved in both calcium influx regulation and mitogenesis.
Moreover, through PA generation, Ang-II stimulates NADH and NADPH
oxidase activity in VSMCs, thus promoting superoxide
generation(39) . Finally, it has been shown that Ang-II has a
strong inducing effect on the release of arachidonic acid from cultured
cardiomyocytes and that arachidonic acid and inositol phosphate
production occurs through distinct Ang-II type 1 and type 2 receptors
and independent signal transduction pathways involving phospholipase C
and phospholipase A
, respectively(21) .
Despite
the increasing knowledge of the signaling pathways stimulated by Ang-II
in different cell lines, the cross-talk between different second
messengers and their correlation with specific physiological responses (i.e. vasoconstriction, hypertrophy, and hyperplasia) remains
relatively unexplored. In this study we demonstrate that in myogenic
cells, ROI generation plays a role in the intracellular transduction of
Ang-II signals for immediate early c-fos and c-jun gene induction, cell proliferation, and hypertrophy. In both
quiescent C2C12 myoblasts and differentiated C2C12 myotubes, Ang-II
significantly stimulates AP1-driven transcription in C2C12 TRE-tk-CAT
stable transfectants and AP1 binding. Using a set of different protein
kinase inhibitors we show that the Ang-II-induced c-Jun/c-Fos
heterodimer binding increase is not mediated by the cAMPdependent
pathway and that protein kinase C and tyrosine kinases are involved.
Moreover, in quiescent C2C12 myoblasts Ang-II induction of both AP1 DNA
binding activity and DNA synthesis is abolished by antioxidants. This
strongly suggests a role for ROIs in the intracellular transduction of
Ang-II signals for both immediate early gene induction and cell
proliferation. Eukaryotic cells continuously produce the ROI
HO
, superoxide
(O
), and hydroxyl radical
(OH
) as by-products of electron transfer
reactions(37) . A condition of oxidative stress, characterized
by above normal levels of ROIs, frequently occurs in cells exposed to
UV light, gamma rays, or low concentrations of H
O
but also upon cell stimulation with cytokines and natural ligands
of other cell surface receptors(40) . Although very high levels
of ROIs, as produced by stimulated neutrophils, are strictly cytocidal
and serve primarily to kill parasites in the organism, the increase of
ROI levels observed in many conditions seems to induce many early
growth signals, including a rise in intracellular pH(41) , the
expression of c-fos, c-jun, and c-myc proto-oncogenes, and the activation of transcription
factors(40, 42, 43) , protein
kinases(44) , protein phosphatases(45) , and ion
channels(46) . A role for oxidative stress has been proposed in
different pathological conditions, such as atherogenesis and
carcinogenesis(47) . Increased concentrations of active oxygen
species have also been measured during the inflammatory stage of the
restenosis process in response to angioplasty(48) . Hyperplasia
is an important aspect of these pathological conditions, and our
results indicate that ROI generation mediate Ang-II mitogenic effects
on quiescent C2C12 cells. Although in the induction of cell
proliferation by growth factors, serum and TPA AP1-activity is required (49) , in C2C12 myotubes Ang-II-dependent AP1 activation occurs
independently from DNA synthesis stimulation. This suggests that in
differentiated cells modulation of TRE-containing genes by AP1 might be
important for other cellular responses. Our results support the
hypothesis that in differentiated C2C12 myotubes Ang-II hypertrophic
effects (i.e. increase of protein synthesis without DNA
synthesis) are also mediated by the generation of ROIs. Thus, ROIs
generation might represent a common second messenger involved in the
induction of both early events (i.e. AP1 binding induction)
and long term metabolic effects (hyperplasia or hypertrophy) in
response to a single growth factor.