© 1999 American Heart Association, Inc.
Vascular Biology |
Angiotensin II Stimulates Collagen Synthesis in Human Vascular Smooth Muscle Cells
Involvement of the AT1 Receptor, Transforming Growth Factor-ß, and Tyrosine Phosphorylation
From the John P. Robarts Research Institute, Vascular Biology Group, and London Health Sciences Centre, Departments of Medicine (Cardiology), Biochemistry, and Medical Biophysics, University of Western Ontario, London, Ontario, Canada.
Correspondence to J. Geoffrey Pickering, London Health Sciences Centre, 339 Windermere Rd, London, Ontario, Canada N6A 5A5. E-mail gpickrng{at}rri.on.ca
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Abstract—Angiotensin II is an established regulator of vascular tone and smooth muscle cell (SMC) growth. However, there are little data about its effect on collagen synthesis by SMCs and none regarding the mechanism of such an effect. We studied the effect of angiotensin II on collagen production by human arterial SMCs, using uptake of [3H]proline into collagenase-digestible proteins, and by ribonuclease protection assay for mRNA encoding the pro
1 chain of type I collagen, the major collagen in arteries. This
revealed a dose-dependent increase in relative collagen synthesis rate
and a dose-dependent increase in pro1(I) collagen mRNA abundance,
with the half-maximal effect at 1.7 nmol/L. Angiotensin
II–stimulated collagen expression was associated with a 6-fold
increase in transforming growth factor-ß (TGF-ß) production
and was inhibited by a neutralizing antibody to TGF-ß. Both collagen
production and TGF-ß release were inhibited by the
AT1-specific antagonist, losartan, but
not by the AT2 receptor antagonist, PD123319.
To determined if tyrosine phosphorylation was
functionally linked to collagen synthesis, we studied the effect of 2
mechanistically distinct inhibitors of tyrosine kinase,
genistein, and tyrphostin A25. These inhibitors abrogated
angiotensin II–mediated procollagen mRNA expression and
angiotensin II–mediated TGF-ß production,
whereas the inactive homolog tyrphostin A1 had no effect. We conclude
that angiotensin II stimulates collagen production
in human arterial SMCs via the AT1 receptor and
an autocrine loop of TGF-ß, induction of which requires tyrosine
phosphorylation.
Key Words: angiotensin • smooth muscle cell • collagen • tyrosine kinase • transforming growth factor
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Introduction |
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Accumulation of interstitial collagen is a structural hallmark of vascular disease. Collagen can occupy most of the volume of occlusive atherosclerotic plaque1 and is a major constituent of human restenosis lesions that develop after angioplasty.2 Increased vascular collagen is also found in hypertension and probably contributes to the increased arterial stiffness of this condition.3 4
The production of collagen in the vessel wall is likely
regulated by a combination of mechanical forces and soluble growth
factors. Of the latter, transforming growth factor-ß (TGF-ß) is
known to stimulate synthesis of collagen in vascular smooth muscle
cells (SMCs),5 whereas interferon-
, fibroblast growth
factor-2, and nitric oxide have been shown to decrease SMC collagen
production.5 6 7
Angiotensin II, long recognized as a regulator of vascular tone, has also garnered interest because of its capacity to act as a vascular cell growth factor. For example, SMCs respond to angiotensin II by increasing protein synthesis,8 9 10 by stimulating DNA synthesis with or without cell proliferation,11 12 13 and by expressing c-fos, c-jun, and c-myc.14 15 16 Although less well studied, angiotensin II may also regulate vascular collagen metabolism. Angiotensin II was found to stimulate collagen synthesis in mesangial cells,17 18 which are functionally similar to SMCs, and angiotensin II has been reported to increase collagen production in rat, chick embryo, and porcine arterial SMCs.19 20 21 The concentration of angiotensin II required for an effect in some of these studies was high,19 20 and possibly not physiological; however, animal data lend support to a potential role for angiotensin II on vascular collagen accumulation. Inhibition of angiotensin-converting enzyme activity suppressed the accumulation of collagen in arteries of growing rats22 and in spontaneously hypertensive rats.23 Recently, infusion of angiotensin II into rats was shown to induce aortic fibrosis.24 These studies thus highlight a relationship between angiotensin II and collagen metabolism, although the mechanism is poorly defined.
Angiotensin II interacts with 2 major subtypes of
receptors, designated AT1 and
AT2, which belong to the superfamily of receptors
with 7 transmembrane domains.25 26 27 The best-characterized
signal transduction pathway is the activation, by the
AT1 receptor, of guanine nucleotide
protein (G protein)-coupled phospholipase C-ß, which in turn
liberates inositol trisphosphate and calcium.28 Recently,
angiotensin II has also been found to initiate signaling
cascades that are similar to those activated by receptor
tyrosine kinases. For instance, angiotensin II
activates the mitogen-activated protein (MAP) kinase
cascade29 30 31 and can stimulate tyrosine
phosphorylation of phospholipase C-,32
signal transducers and activators of transcription
(STATs),33 p125FAK,34
and paxillin.35 36 Evidence is emerging that the
contractile and hypertrophic actions of angiotensin II are
at least partly mediated by tyrosine kinase–dependent signaling
pathways.37 38 This participation of tyrosine
phosphorylation–mediated signaling events downstream
to a G protein–coupled receptor both highlights the diversity of
signaling cascades that can be initiated by angiotensin II
and brings new opportunities for therapeutic control of vascular
remodeling.
The biochemical pathway by which angiotensin II controls collagen production in SMCs is unknown. The transmembrane signal transduction pathway is obscure and it is likewise not known if the effect of angiotensin II on collagen production is direct or via the release of other fibrogenic factors.39 Accordingly, the purpose of this study was (1) to determine if angiotensin II stimulates the elaboration of collagen by human vascular SMCs; (2) to clarify the membrane receptor subtype that mediates angiotensin II–stimulated collagen expression; (3) to determine whether TGF-ß is involved in the fibrogenic response to angiotensin II; and (4) to investigate the role of tyrosine kinases in angiotensin II–mediated collagen production. Our findings provide novel evidence that angiotensin II initiates a tyrosine kinase–mediated fibrogenic cascade in human SMCs.
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Methods |
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Reagents
All culture media and supplements, as well as genistein were purchased from Life Technologies. Human angiotensin II, sodium ascorbate, and bacterial collagenase were purchased from Sigma Chemical Co. Tyrphostin A25 and tyrphostin A1 were purchased from Calbiochem. Anti-smooth muscle
-actin antibody (clone 1A4) was
obtained from Dako. Neutralizing monoclonal antibody to TGF-ß1 and
TGF-ß2 was obtained from Genzyme. Human fibronectin was isolated from
human plasma by gelatin-Sepharose
chromatography.40
[32P]dUTP and
L-[3H]proline were obtained from
Dupont/NEN and [3H]thymidine from ICN.
Losartan was generously supplied by W. Henkler, Merck and Co,
Inc, and the AT2 receptor antagonist
PD123319 was purchased from Research Biochemical International. Human
platelet TGF-ß1 was obtained from R & D Systems.
Smooth Muscle Cell Culture
Primary cultures of human arterial SMCs were
initiated by explant outgrowth from unused segments of internal
thoracic artery retrieved at the time of coronary artery bypass
surgery, as previously described.41 42 The identity of
vascular SMCs was confirmed morphologically and by positive
immunostaining for smooth muscle -actin. Cells were
grown on fibronectin-coated culture dishes (10
µg/cm2) and cultured in media (M199)
supplemented with 100 U/mL penicillin G, 100 µg/mL streptomycin, and
the designated concentration of FBS. All experiments were performed by
using human SMCs in the third or fourth subculture.
Collagen Synthesis Assay
Human arterial SMCs at near confluence were rendered
quiescent in media supplemented with 1% FBS for 24 hours and
subsequently stimulated with the designated concentration of
angiotensin II and sodium ascorbate (10 µg/mL) for 48
hours. Control cultures were incubated with ascorbate plus vehicle.
SMCs were pulsed for the last 20 hours with 15 µCi/mL
L-[3H]proline (100 mCi/mmol) in
proline-free DMEM. During this period the media was supplemented with
1% dialyzed FBS, fresh sodium ascorbate (10 µg/mL), and
ß-aminoproprionitrile (80 µg/mL).
The relative rate of collagen synthesis was measured according to
Kolpakov et al7 and Kulik and Alvarado.43 In
brief, medium was mixed with buffer containing 0.65 mol/L NaCl,
100 mmol/L Tris-Cl, pH 7.4, 4.7 mmol/L
CaCl2, 1.25 mg/mL n-ethyl maleimide,
and 50 µg/mL BSA. Samples were split into 2 equal portions and highly
specific collagenase (Sigma type VII, 10 U/mL) was added to
1 portion. The samples were incubated for 90 minutes at 37°C and
undigested proteins precipitated on ice with 10% trichloroacetic
acid. Washed pellets were air-dried, dissolved in 0.1 N NaOH,
and radioactivity incorporated into trichloroacetic acid–precipitable
counts was measured by liquid scintillation counting. The rate of
collagen synthesis, relative to that of all proline-containing
proteins, was calculated assuming that the number of proline residues
in collagen is 5.4-fold higher than that in noncollagen
proteins.44 The relative rate of collagen synthesis
was thus determined as:
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RNA Isolation and Ribonuclease Protection Assay for pro1(I)
Collagen mRNA
SMCs were stimulated with various concentrations of
angiotensin II, in the presence or absence of
angiotensin II receptor antagonists,
anti-TGF-ß antibody, and tyrosine kinase inhibitors.
After incubation of SMCs under the designated conditions, cells were
lysed in a solution of 4 mol/L guanidinium isothiocyanate, 25
mmol/L sodium citrate, pH 7.0, 0.1 mol/L 2-mercaptoethanol and 0.5%
Sarkosyl. Total RNA was isolated by using the acid-phenol
technique.45 Abundance of pro1(I) collagen mRNA was
quantified by ribonuclease (RNase) protection assay. A cDNA fragment of
human pro1(I) collagen (pSP3, gift of Dr C. Farrell, Amgen Inc,
Thousand Oaks, California) served as the template from which a
300-base radiolabeled riboprobe was synthesized. The template was
linearized with XhoI and 0.5 µg of template was incubated
with T7 RNA polymerase and [32P]dUTP (800
Ci/mmol), as per the manufacturer's instructions (Promega). Specific
activity of the probe was >108 cpm/µg. Labeled
riboprobe (120 pg) was added to 2.0 to 3.0 µg of total RNA from each
experimental sample. Hybridization was allowed to proceed at 63°C for
16 hours in the presence of 80% formamide, 40 mmol/L HEPES, pH
6.7, 0.4 mol/L NaCl, 1 mmol/L EDTA, and 0.24 µg/µL transfer
RNA. The samples were then incubated with RNase A (50 mU/µg) and
RNase T1 (34 U/µg) for 1 hour at 34°C to degrade single-stranded
RNA. The remaining double-stranded RNA was precipitated with 50 µg of
herring sperm DNA and cold 20% trichloroacetic acid, filtered through
glass-fiber filters, and counted in scintillation cocktail. The amount
of pro1(I) collagen mRNA per sample was determined from a standard
curve, generated from hybridization reactions by using known
concentrations of a pro1(I) collagen mRNA 300-base fragment. The
latter was derived by transcribing the cDNA template, using SP6 RNA
polymerase, and quantifying the transcript by UV spectrophotometry. The
result was multiplied by 17.7 [=(5800+4800)/(2x300)], recognizing
that the full-length pro1(I) collagen mRNA consists of 2 transcripts
of roughly equal abundance, of size 5.8 and 4.8 kb. Expression of human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
mRNA (pHcGAP, American Type Culture Collection [ATCC]) was also
quantified by RNase protection assay, and abundance of pro1(I)
collagen mRNA was expressed relative to that of GAPDH.
Quantification of TGF-ß
Quiescent SMCs (1% FBS) were stimulated with
angiotensin II for 48 hours in the presence or absence of
protein tyrosine kinase inhibitors or their inactive
homologs. Angiotensin II at both
10-8 and 10-7 mol/L were
used for these studies, as these concentrations were determined to
stimulate plateau or near-plateau rates of collagen synthesis. Washed
cells were lysed and total protein content determined by Lowry assay
(Bio-Rad). The quantity of TGF-ß in conditioned media was determined
by mink lung epithelial cell growth inhibition
assay.46 47 48 In brief, CCL-64 mink lung cells (ATCC) were
maintained in DMEM supplemented with 10% FBS, 10 mmol/L HEPES,
and 20 mmol/L glutamine. Cells were plated onto 96-well plates
(2x104 cells per well) in 100 µL of the
above-described medium and allowed to adhere for 2 hours after which
100 µL of conditioned medium, or TGF-ß1 control solution, was added
to the wells. Cells were incubated for 21 hours and then pulsed with
[3H]thymidine (5 µCi/mL, 76 Ci/mmol, ICN) for
4 hours. Thymidine incorporation was determined by automated harvesting
(Tomtec) of cells onto glass-fiber filters and counting (Trilax 1450
MicroBeta counter). To assess total TGF-ß (active plus latent),
latent TGF-ß was activated by acidification with HCl to pH
1.5 for 1 hour, followed by neutralization with NaOH. TGF-ß
concentration was determined from the standard curve generated by using
purified human platelet TGF-ß, and expressed relative to protein
content of the cell lysate.
Statistics
Data are expressed as mean±SEM values. Comparisons were made by
t test or ANOVA with Scheffé's post hoc test.
Statistical significance was set at P<0.05.
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Results |
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Angiotensin II Stimulates Collagen Synthesis in Human SMCs
We examined the effect of angiotensin II on the synthesis and secretion of collagen by human SMCs by metabolically labeling cells with [3H]proline and quantifying proline incorporation into collagenase-digestible proteins. As illustrated in Figure 1
, the relative
rate of collagen synthesis was increased by angiotensin II
in a dose-dependent fashion, with a statistically significant increase
at 10-8 and 10-7 mol/L
angiotensin II. Time course analysis indicated no
detectable increase in collagen production 5 or 10 hours after
stimulation with angiotensin II, an intermediate effect at
24 hours, and plateau levels by 48 hours (data not shown).
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Angiotensin II Stimulates Expression of pro1(I)
Collagen mRNA in Human SMCs
To assess the effect of angiotensin II on type 1
procollagen gene expression, RNA was harvested after 48 hours of
stimulation with angiotensin II. To accurately quantify
procollagen mRNA abundance, we established an RNase protection assay
for pro1(I) collagen mRNA. A standard curve was generated from
hybridization reactions by using known amounts of pro1(I) collagen
mRNA, and was used to quantify absolute concentrations of pro1(I)
collagen mRNA and to confirm that the experimental results fell within
the linear range of the assay. As shown in Figure 2A, stimulation with
angiotensin II yielded a potent, dose-dependent increase in
pro1(I) collagen mRNA with a half-maximal effect at 1.7 nmol/L.
GAPDH levels were unaffected by angiotensin II.
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P<0.05 versus SMCs
treated with angiotensin II alone.
Angiotensin II Stimulates Collagen Expression via the
AT1 Receptor
The rise in pro1(I) collagen mRNA abundance was found to
reflect that for relative collagen synthesis rate. Because of this, and
in view of the sensitive quantification afforded by the RNase
protection assay, we used procollagen mRNA abundance, relative to that
of GAPDH mRNA, as an end point to characterize the relevant
transmembrane signaling events. To establish the cell surface receptor
mediating the response, SMCs were incubated with losartan,
which acts as a competitive inhibitor of the
AT1 receptor, or the AT2
receptor antagonist PD123319, each added 30 minutes before
addition of 10-8 mol/L angiotensin
II. As shown in Figure 2B
, neither losartan
(10-7 mol/L) nor PD123319
(10-7 mol/L) affected basal expression of
pro1(I) collagen mRNA. However, angiotensin
II–stimulated procollagen mRNA expression was inhibited by the 10-fold
molar excess of losartan but not by the same concentration of
PD123319; 100-fold excess of the respective inhibitors
showed similar results (data not shown).
Angiotensin II–Stimulated Collagen Expression Is
Mediated by Autocrine TGF-ß
Angiotensin II may influence cell function by inducing
the expression of growth factors, which then act on the cell in an
autocrine fashion.12 39 Extrapolating from observations in
other experimental systems,18 we considered that
angiotensin II might stimulate collagen indirectly,
specifically by inducing the expression of TGF-ß. To determine if
stimulation of SMCs with angiotensin II resulted in
elaboration of TGF-ß, a mink lung epithelial cell TGF-ß bioassay
was used. As shown in Figure 3, 48 hours
of treatment with either 10-8 mol/L or
10-7 mol/L angiotensin II increased
total TGF-ß (latent plus active) over basal levels by 
6-fold
(P<0.001). The proportion of active TGF-ß in the culture
media was 3.4% and 2.6% of total TGF-ß in control and
angiotensin-treated cultures, respectively. Incubation of
SMCs with angiotensin II increased the amount of active
TGF-ß in the culture media by 4-fold (P<0.001). As
with the expression of pro1(I) collagen mRNA, production of
TGF-ß by angiotensin II was inhibited by
losartan, but not by PD123319, implicating the
AT1 receptor in this response (Figure 3B).
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We next determined if angiotensin II–stimulated
production of TGF-ß mediated the observed increase in
collagen expression in SMCs. This was performed by incubating cells
with angiotensin II together with a monoclonal anti-TGF-ß
antibody that neutralizes the biological activity of TGF-ß1 and
TGF-ß2 (1D11.16, Genzyme).49 Pro1(I) collagen mRNA
was quantified by RNase protection assay. Figure 4 shows that the anti-TGF-ß antibody
(30 µg/mL) had no significant effect on basal collagen expression;
however, it significantly inhibited the angiotensin
II–stimulated increase in pro1(I) collagen mRNA. In contrast,
control IgG (P3, 30 µg/mL; ATCC) had no effect on either basal or
angiotensin II–stimulated procollagen expression.
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Angiotensin II–Stimulated Collagen Expression Is
Mediated by Tyrosine Kinase Action
To explore the possibility that tyrosine kinases may be involved
in upregulation of type I collagen by angiotensin II, we
used 2 mechanistically distinct inhibitors of tyrosine
phosphorylation. Genistein inhibits ATP binding to
tyrosine kinases and thus its ability to donate
phosphate.50 Tyrphostins inhibit tyrosine kinases by
competitively binding to the catalytic site of the
enzyme.51 By using RNase protection assay, we first
established the effect of a 48-hour exposure to various concentrations
of genistein and tyrphostin A25, in the absence of
angiotensin II, on expression of pro1(I) collagen mRNA.
This allowed us to define an upper dose for each tyrosine kinase
inhibitor that would not impair basal human SMC function
vis-à-vis collagen expression; this proved to be 10 µmol/L
for genistein and 50 µmol/L for tyrphostin A25. Subsequently,
quiescent SMCs were treated with these doses of inhibitor
for 40 minutes followed by stimulation with angiotensin II
in the presence of inhibitor for a further 48 hours. As
illustrated in Figure 5, genistein
significantly inhibited angiotensin II–mediated pro1(I)
collagen mRNA expression. In similar manner, tyrphostin A25, but not
the inactive homolog tyrphostin A1, blocked angiotensin
II–stimulated procollagen expression.
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Tyrosine Kinase Action During Angiotensin II–Mediated
Collagen Production Is Localized Upstream of TGF-ß
Expression
Given that production of collagen by
angiotensin II was mediated by an autocrine loop of
TGF-ß, 2 potential transmembrane pathways must be considered with
respect to localizing the tyrosine kinase–dependent events. We
speculated that the proximal arm, ie, stimulation of TGF-ß
expression, was tyrosine kinase dependent. To test this, SMCs were
incubated for 48 hours with angiotensin II and either
genistein, tyrphostin A25, or the inactive homolog tyrphostin A1, and
conditioned medium was assayed for TGF-ß activity. As shown in Figure 6, 10 µmol/L genistein as well as
50 µmol/L tyrphostin A25 significantly inhibited
angiotensin II–stimulated TGF-ß production,
whereas 50 µmol/L tyrphostin A1 had no effect. Direct addition
of either tyrosine kinase inhibitor to epithelial cell
cultures showed no effect on DNA synthesis rates of the epithelial
cells (data not shown).
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Although the mechanism of angiotensin II–mediated SMC contraction has been intensively studied, much less is known about the chronic trophic changes induced by angiotensin II. Vascular SMC collagen synthesis is critical to vascular remodeling2 52 53 and previous studies have suggested that angiotensin II may stimulate SMC collagen production.19 20 21 The mechanism of such an effect, however, has been unknown. In these studies, we have demonstrated that angiotensin II stimulates collagen synthesis in human arterial SMCs. The response is associated with expression of mRNA encoding the pro
1(I) chain of type I collagen, the major
collagen species found in the normal and diseased artery wall. We
further found that the AT1 receptor was
responsible for this action of angiotensin II, and that
stimulation of this receptor initiated an autocrine loop of TGF-ß
expression and stimulation that, in turn, was responsible for the
angiotensin II–mediated collagen production.
Moreover, the studies establish a novel link between
angiotensin II–mediated collagen production and
tyrosine phosphorylation. Specifically, we observed
that tyrosine kinase activity is required for induction of the
autocrine TGF-ß loop by angiotensin II. In the current study, losartan inhibited angiotensin II–mediated collagen expression, whereas PD123319 did not, implicating a role for the AT1 receptor subtype in this response. The AT1 receptor is a 7-transmembrane receptor that is responsible for most of the physiological actions of angiotensin II. The finding that collagen synthesis in human SMCs is also regulated by this receptor is noteworthy in view of reports of an association between AT1 polymorphism and aortic stiffness in hypertension.54 Thus, AT1 receptor–mediated collagen production may be a key determinant of the mechanical properties of the vessel wall. Several studies of angiotensin II receptor binding in human arterial SMCs have documented the presence of the AT1 receptor but the absence of the AT2 receptor,55 56 57 and our results are therefore consistent with these receptor data. However, the findings would appear to contrast with a provocative study by Levy et al,24 which showed that angiotensin II–induced fibrosis of the rat aortic media was blocked by the AT2 receptor antagonist PD123319. Because collagen synthesis and/or collagen gene expression were not evaluated in the study by Levy et al,24 the extent to which the observed fibrotic response reflected increased collagen production versus decreased collagen degradation is unknown. As well, the presence of AT2 receptors in rat aortic medial SMCs has not been directly established and it is conceivable that other cells might have been involved. Endothelial cells can express AT2 receptors58 and could potentially modulate collagen metabolism by SMCs, possibly through angiotensin II–mediated effects on nitric oxide production.7 59 Finally, differences between rat and human SMC responses to angiotensin II must be considered in view of the discrepant findings of pharmacological disruption of the renin–angiotensin axis on vascular remodeling in rats and humans (for review, see Pratt and Dzau60 ).
It has been increasingly recognized that angiotensin II may
exert at least some of its growth factor-like effects by inducing the
secretion of growth factors from SMCs, which can then act in an
autocrine fashion. In rat aortic SMCs, angiotensin II has
been reported to increase expression of TGF-ß,12 39
platelet-derived growth factor A-chain,16 and
fibroblast growth factor-2.39 Because TGF-ß is a potent
stimulator of collagen synthesis in SMCs5 and probably
involved in the genesis of several fibrotic disorders, we considered
that the angiotensin II–mediated production of
collagen by human SMCs may be mediated by TGF-ß. Our data established
that angiotensin II stimulated the release of a substantial
amount of TGF-ß from human SMCs, and that TGF-ß in the
activated form increased in the media by 400%. The absolute
amount of active TGF-ß found in the media (0.5 to 0.6 ng/mL) was
within the concentration range previously shown to stimulate collagen
synthesis in human SMCs,5 and within the range that we
have observed leads to increased expression of pro1(I) collagen mRNA
(data not shown). Furthermore, angiotensin II–mediated
collagen expression was blocked by a neutralizing antibody to
TGF-ß1/TGF-ß2, establishing that the production of
bioactive TGF-ß was a mechanistic pathway for the increase in
collagen expression. In addition to mediating angiotensin
II–induced collagen production in SMCs, autocrine TGF-ß has
also been suggested to modulate the proliferative and migratory
response of SMCs to angiotensin II.12 61
Release of TGF-ß because of activation of the
AT1 receptor may thus be an important means by
which angiotensin II acts as a multifunctional growth
factor to control vascular structure.
The AT1 receptor lacks intrinsic tyrosine kinase
activity. Nevertheless, there is now substantial evidence that, when
activated, this receptor induces tyrosine
phosphorylation of cytoplasmic second-messenger
proteins. Angiotensin II has been shown to directly
activate the protein tyrosine kinase
pp60c-src62 and several substrates of
pp60c-src are phosphorylated on
tyrosine after angiotensin II stimulation, including
phospholipase C-1, pp120, pp125FAK, and
paxillin.34 35 63 Tyrosine phosphorylation
and activation of the JAK–STAT signaling cascade have also been shown
to be a direct response to AT1 receptor
stimulation by angiotensin II.33 64 Recently,
angiotensin II has also been demonstrated to
activate the Ras pathway, via the actions of the tyrosine
kinase pp60c-src.65
Despite major progress in understanding the tyrosine
phosphorylation cascades induced by
angiotensin II, relatively little is known about the
functional consequences of these actions. We used 2 different
inhibitors of tyrosine kinase to study the role of protein
tyrosine phosphorylation on angiotensin
II–stimulated collagen synthesis. Genistein inhibits the binding of
ATP to tyrosine kinases and its use has previously suggested that
tyrosine kinase pathways modulate angiotensin II–induced
calcium transients in SMCs.66 Tyrphostins bind the
substrate binding site of the protein tyrosine kinase domain, and may
be more specific for tyrosine kinases than genistein. These agents have
been used recently to demonstrate that angiotensin
II–mediated SMC growth requires tyrosine
phosphorylation.37 38 Our data revealed
that both genistein and tyrphostin A25 inhibited
angiotensin II–mediated expression of pro1(I) collagen
by human SMCs at concentrations that did not impair basal collagen
expression. The observation that under conditions of stable basal
collagen production, these biochemically distinct
inhibitors produced a similar effect strongly supports a
role for tyrosine phosphorylation in
angiotensin II–mediated collagen production by
arterial SMCs.
In view of our observation that angiotensin II stimulates
collagen expression indirectly, ie, via elaboration of TGF-ß, there
are 2 broad pathways that potentially could involve tyrosine
phosphorylation, angiotensin II–induced
production of TGF-ß or TGF-ß–induced production of
collagen (or both). We established that the initial limb is a tyrosine
kinase–dependent process, because both genistein and tyrphostin A25,
but not the inactive homolog tyrphostin A1, blocked the
production of TGF-ß by angiotensin II. We
speculate that this proximal arm of the fibrogenic cascade is unique in
its requirement for tyrosine kinase action. Based on current
understanding of TGF-ß signaling, there is little support for the
involvement of protein tyrosine phosphorylation in
TGF-ß–induced collagen production. The transmembrane
receptor events in this include interaction of primary and transducing
receptor subtypes, both of which are serine–threonine kinases.
Subsequent propagation of signal is mediated by members of the Smad
protein family, which become phosphorylated on serine
residues.67 In contrast to the signaling cascade initiated
by TGF-ß stimulation, there is supportive evidence that regulation of
TGF-ß expression may require tyrosine kinase activity. Recently,
expression of TGF-ß after arterial injury was shown to be
inhibited by genistein.68 As well, TGF-ß1 gene
transcription in neuronal cells was found to be activated by
ligands of protein tyrosine kinase receptors and this response was
mediated by p21ras.69 The
involvement of Ras in TGF-ß gene expression is particularly
noteworthy in view of recent observations that angiotensin
II can activate the Ras–Raf–MAP kinase pathway in SMCs and
does so in a Src tyrosine kinase–dependent fashion.65
Thus, the site of action of the tyrosine kinase inhibitors
in the present study might be localized between the
AT1 receptor and Ras. A potential pathway
depicting the process of angiotensin II–mediated collagen
production in SMCs is shown in Figure 7.
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In summary, angiotensin II stimulates collagen synthesis in human arterial SMCs. The response is seen at angiotensin II concentrations that are likely relevant to human vascular disease and is consistent with its role as a multifunctional growth factor for SMCs. The effect is mediated by the AT1 receptor, suggesting that pharmacological therapy with AT1 receptor antagonists may modulate collagen accumulation during vascular disease. Furthermore, our data suggest a novel mechanism for this angiotensin II–initiated fibrogenic cascade, whereby tyrosine kinase activity mediates the stimulated production of TGF-ß.
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Acknowledgments |
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This work was supported by grants from the Medical Research Council of Canada (MRC) and the Merck-Frosst Medical Schools Grants Program. J.G.P was supported by an MRC Scholarship and a Career Investigator Award from the Heart and Stroke Foundation of Ontario. S.L. was supported by a Robarts Research Institute-Merck Frosst Fellowship in Medical Research.
Received March 31, 1998; accepted January 14, 1999.
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