© 1997 American Heart Association, Inc.
Articles |
Increased Pressure Induces Sustained Protein Kinase C–Independent Herbimycin A–Sensitive Activation of Extracellular Signal–Related Kinase 1/2 in the Rabbit Aorta in Organ Culture
From INSERM U-141 and IFR Circulation, Hôpital Lariboisière (S.L., A.A.B., R.M., A.T.), Paris, France, and the Laboratory of Molecular Endocrinology, Cardiology Research Center (K.G.B., V.A.T.), Moscow, Russia.
Correspondence to Alain Tedgui, INSERM U 141, 41 Bd de la Chapelle, 75475 Paris Cedex 10, France. E-mail tedgui{at}infobiogen.fr
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Abstract The 42- and 44-kD mitogen-activated protein kinases, also referred to as extracellular signal–related kinase (ERK) 2 and 1, respectively, may be transiently activated by stretching vascular smooth muscle cells (VSMCs). Using an organ culture model of rabbit aorta, we studied short- and long-term ERK1/2 activation by intraluminal pressure (150 mm Hg). Activation of ERK1/2 was biphasic: it reached a maximum (217.5±8.4% of control) 5 minutes after pressurizing and decreased to 120.7±5.1% of control after 2 hours. Furthermore, after 24 hours of pressurizing, ERK1/2 activity was as high (241.8±14.7% of control) as in the acute phase. Long-term pressure-induced ERK1/2 activation correlated with stimulation of tyrosine phosphorylation of proteins in the 125- to 140-kD range. Neither protein kinase C inhibitors (1 µmol/L staurosporine or 50 µmol/L bisindolylmaleimide-I) nor tyrosine kinase inhibitors (50 µmol/L tyrphostin A48 or 50 µmol/L genistein) affected pressure-induced ERK1/2 activation. However, the Src-family tyrosine kinase inhibitor herbimycin A (500 nmol/L) did reduce both 5-minute (by 92±8%) and 24-hour (by 63±7%) pressure-induced ERK1/2 activation. Thus, our results demonstrate a sustained activation of ERK1/2 and tyrosine kinases by intraluminal pressure in the arterial wall. Pressure-induced ERK1/2 activation is PKC independent and Src-family tyrosine kinase dependent and possibly includes activation of extracellular matrix–associated tyrosine kinases.
Key Words: mechanical stimulation • protein kinase C • signal transduction • extracellular signal–related kinase • tyrosine kinase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Elevated blood pressure in hypertension results in vascular remodeling, characterized by increased vessel wall thickness, VSMC hypertrophy and/or hyperplasia, and accumulation of extracellular matrix.1–3 The precise mechanisms controlling these processes remain to be elucidated. However, increased mechanical distension and/or compression of VSMCs during the acute phase of hypertension is hypothesized to trigger hypertension-induced vascular remodeling.2,4
Many signaling systems are activated in response to single or short-term cellular stretch. Stretching cultured cells leads to the opening of stretch-activated ionic channels5,6 and to activation of phosphoinositide turnover,7 PKC,8 and Ca2+/calmodulin-dependent kinases.9 Activation of these pathways leads to immediate cellular responses to stress, such as secretion of bioactive substances,10,11 activation of smooth muscle contraction,12 and cytoskeletal rearrangement.13 However, these intracellular events occur within the first 15 to 20 minutes after the onset of mechanical stimulation and cannot explain the sustained increase in extracellular matrix production and VSMC hypertrophy, which characterize hypertension-induced vascular remodeling.
The ERK cascade is a novel signaling pathway possibly involved in mechanotransduction.8,14,15 ERK 1 and 2, identified as 44- and 42-kD MAP kinases, respectively, are regulatory kinases convergent for signals transduced by many pathways, and they participate in the control of gene expression. Generally, activation of the ERK1/2 cascade is associated with activated total tyrosine phosphorylation.16,17 ERK1/2 is able to activate the ternary complex formation factor p62TCF/elk-1.18,19 ERK1/2 induces de novo synthesis of immediate response genes such as c-fos.19,20 ERK1/2 is also able to stimulate total protein synthesis via interruption of PHAS-I/eIF4-BP complex formation.14 As recently shown,21 ERK1/2 is activated in vivo during acute experimental hypertension. Moreover, stretch of the vessel segments in vitro also induces ERK1/2 activation.15
The objectives of the present study were as follows: (1) to verify whether ERK1/2 is activated during long-term mechanical stimulation of vascular segments and (2) to study the interrelationship between activation of tyrosine kinases and ERK1/2 on mechanical stimulation using an organotypic model of rabbit aorta. ERK1/2 activity was assessed in vessel segments pressurized and perfused at different regimens for up to 3 days.
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Materials and Methods |
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Artery Preparation
Aortas were prepared for organ culture as previously described.22 Briefly, male New Zealand White rabbits (2 to 2.5 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV). The animals were intubated and artificially ventilated. The abdominal and pleural cavities were opened, and the entire length of the descending thoracic aorta was exposed. The intercostal arteries were cauterized 2 to 3 mm from the vessel, which was cleaned of adhering periadventitial tissue. A cannula was inserted retrogradely into the distal end of the aorta and connected to a reservoir placed 100 cm above the animal. The proximal end of the aorta was then ligated just below the arch, and a second cannula, pointing distally, was inserted anterogradely. This procedure allowed maintenance of constant intraluminal pressure within the artery, preventing collapse of the vessel wall and endothelial damage.22 A ligature was tied around the midregion of the aorta, and a cannula was inserted distally to this ligature. The lower part of the descending thoracic aorta was then excised on splints. A similar procedure was used to excise the upper part of the aorta.
Organ Culture
Each arterial segment was connected to a perfusion
circuit consisting of a three-port glass reservoir, a peristaltic pump
(Masterflex 60648, Cole-Palmer Instrument Co), and a pressure chamber,
which permitted the application of a controlled hydrostatic pressure to
the intraluminal compartment. The two lateral ports of the glass
reservoir were used for input and output of the circulating
intraluminal medium, which was the same as the extraluminal medium
described below. Organ culture of arterial segments was
carried out under sterile conditions in an incubator containing 5%
CO2 at 37°C. Aortic segments were immersed in an organ
culture bath filled with DMEM containing antibiotics (penicillin, 100
IU/L; streptomycin, 100 mg/L; and amphotericin B, 10
µg/L) and supplemented with 20% FCS (Boehringer
Mannheim). Some acute experiments were conducted in the absence of FCS,
and this was found not to affect ERK1/2 activation by intraluminal
pressure.
Experimental Protocol
To study activation of the vessel segments by pressure, segments
were cannulated, mounted on the organ culture system, and left at
70 mm Hg for 1.5 hours for equilibration. Thereafter, for 5, 20,
or 120 minutes or for 24 hours an intraluminal pressure of 150
mm Hg, comparable to levels found in hypertension, was applied to
experimental segments; control segments were kept at 10 mm Hg.
In long-term experiments, aortas were maintained in the organ culture system for 1 or 3 days at subphysiological (10 or 50 mm Hg), normal (80 mm Hg), or hypertensive levels (150 mm Hg) and perfused at a low flow of 0.2 mL/min to ensure renewal of culture medium within the aorta but to avoid any shear effects. In an additional set of experiments, vessel rings 3 to 5 mm in length were cultured in Petri dishes at zero transmural pressure to serve as relaxed controls. Furthermore, segments used for comparison of different pressure regimens were obtained from the same rabbit and processed simultaneously.
For all experiments, vessels maintained at low pressure (10 mm Hg) for 5, 20, or 120 minutes or 1 or 3 days served as controls (ERK1/2 activity taken as 100%) for vessels pressurized at 50, 80, or 150 mm Hg during the equivalent time period. From six to eight aortic segments were studied under each experimental condition.
At the end of each experiment, aortic segments were rapidly removed from the perfusion system, briefly washed in ice-cold buffer A (20 mmol/L Tris-HCl, pH 7.5, 5 mmol/L EGTA, 150 mmol/L NaCl, 20 mmol/L glycerophosphate, 10 mmol/L NaF, and 1 mmol/L sodium orthovanadate), and frozen in liquid nitrogen. Frozen segments were stored at -70°C until use for ERK1/2 assays.
To evaluate the role of PKC in ERK1/2 activation, vessel segments were pressurized in the presence of PKC inhibitors staurosporine (1 µmol/L) or B-I (50 µmol/L). To check the potency of these inhibitors, we tested their effects on PDBu-induced contraction of fresh aortic rings at concentrations used for long-term vessel pressurizing. PDBu (1 µmol/L) caused a slowly developing contraction of aortic segments, which reached a maximum after 30 minutes. B-I completely inhibited PDBu-induced contraction, and staurosporine had a 70% inhibitory effect. Vehicle (dimethyl sulfoxide, <0.1%) did not affect basal tyrosine phosphorylation or ERK1/2 activity. Furthermore, these inhibitors blocked PDBu-induced ERK1/2 activation in vessel rings.
Finally, a role for tyrosine kinases in intraluminal pressure–induced ERK1/2 activation was investigated using tyrphostin A48 (50 µmol/L), genistein (50 µmol/L), or herbimycin A (500 nmol/L). Inhibitor concentrations were verified against PDGF-AB (50 ng/mL)–stimulated ERK1/2 activation. Compared with unstimulated rings, aortic ring segments exposed to PDGF displayed an enhanced ERK1/2 activity that was inhibited by the tyrosine kinase inhibitors at the concentrations indicated. PKC or tyrosine kinase inhibitors were added to the culture medium of vessels pressurized at 150 mm Hg for 5 minutes or 24 hours.
Tissue Extraction
Frozen vessel segments were pulverized under liquid nitrogen.
The powders were resuspended in ice-cold lysis buffer B (20
mmol/L Tris-HCl, pH 7.5, 5 mmol/L EGTA, 150
mmol/L NaCl, 20 mmol/L glycerophosphate, 10
mmol/L NaF, 1 mmol/L sodium orthovanadate, 1%
Triton X-100, 0.1% Tween-20, 1 µg/mL aprotinin, 1
mmol/L phenylmethylsulfonyl fluoride, 0.5
mmol/L N-tosyl-L-phenylalanine
chloromethyl ketone, and 0.5 mmol/L
N
-p-tosyl-L-lysine chloromethyl
ketone) at a ratio of 0.3 mL/10 mg wet wt. Extracts were incubated on
ice for 15 minutes and then centrifuged (12 000g
for 15 minutes at 4°C). The detergent-soluble supernatant fractions
were retained, and protein concentrations in samples were equalized
using a Bio-Rad protein assay.
In-Gel ERK1/2 Assays
Kinase assays in MBP-containing gels were performed as described
previously23 with minor modifications. Tissue samples were
lysed with buffer B. Laemmli24 sample buffer (70 µL) was
added to 100 µL aliquots, and samples were boiled for 3 minutes and
loaded on a 9% SDS-polyacrylamide gel containing 0.5
mg/mL MBP. The SDS-PAGE was performed using a Bio-Rad
Mini-Protean device. After electrophoresis was finished, SDS was
removed from the gel by washing with three changes of 80 mL each of
20% 2-propanol in 50 mmol/L Tris-HCl (pH 8.0) for 15
minutes and then with 100 mL of 50 mmol/L Tris-HCl (pH 8.0)
containing 5 mmol/L 2-mercaptoethanol for 30 minutes at
room temperature. Gels were further treated with 50 mL of 6
mol/L guanidine-HCl in 50 mmol/L Tris-HCl (pH 8.0)
at room temperature for 1 hour, which was followed by five changes of
50 mmol/L Tris-HCl (pH 8.0) containing 0.04% Tween-20 and
5 mmol/L 2-mercaptoethanol at room temperature for 15
minutes each, and left in the same buffer at +4°C overnight.
Afterward, gels were immersed in 10 mL of 40 mmol/L HEPES
(pH 8.0) containing 2 mmol/L dithiothreitol and 10
mmol/L MgCl2 for 1 hour at 25°C.
Phosphorylation of MBP was carried out by incubating
the gels with 50 µCi [
-32P]ATP at 25°C for 3 hours
in 10 mL of 40 mmol/L HEPES (pH 8.0) containing 2
mmol/L dithiothreitol, 10 mmol/L MgCl2,
0.5 mmol/L EGTA, and 40 µmol/L ATP. After
incubation, the gels were washed with 10% acetic acid and 1% sodium
pyrophosphate until the radioactivity of the washing solution became
negligible. The washed gels were dried and then subjected to
autoradiography.
Immunoblotting
Lysates containing equal amounts of protein (30 µg) were
electrophoresed on a 9% polyacrylamide gel and transferred to
nitrocellulose membranes. Membranes were blocked with 5% nonfat dry
milk in TBST (20 mmol/L Tris, pH 8.0, 150
mmol/L NaCl, and 0.1% Tween-20) for 1 hour and were then
incubated with anti-ERK1 or PY20 monoclonal antibodies at a dilution of
1:1000 in TBST for an additional hour. An enhanced chemiluminescence
system was used as the detection method. Blots were washed and
subjected to autoradiography. Molecular weights of
proteins were estimated by using prestained markers (Bio-Rad,
161-0324). Each lane presented in a single panel of the gel
picture was from the same gel and the same exposure of the
autoradiogram, although in some cases lanes were cut
for the final figure production.
Materials
All culture reagents were purchased from GIBCO-BRL.
[-32P]ATP (6000 Ci/mmol), nitrocellulose membranes,
horseradish peroxidase–conjugated anti-mouse secondary antibodies, and
the enhanced chemiluminescence detection system were obtained from
Amersham. Gel electrophoresis reagents were from Bio-Rad.
Staurosporine and bisindolylmaleimide-1 came from LC
Laboratories Europe. Monoclonal anti-ERK1 antibody and
anti-phosphotyrosine antibody (PY20) were purchased from Transduction
Laboratories. MBP, PDBu, and all other chemicals were obtained from
Sigma Chemical Co.
Data Analysis
For in-gel kinase assays, percent activation of ERK1/2 was
expressed relative to both protein content of samples tested or to
autoradiographic density of ERK1/2 in corresponding Western
blots. Results of both variants of estimation were highly
consistent. For Western blot analysis,
autoradiograms exposed in the linear range of film
density were scanned (Studioscan, AGFA), and densitometric
analysis was performed with NIH Image 1.49 software. All
experiments were performed at least three times, and results are
expressed as mean±SE. A one-way ANOVA was constructed with data of
ERK1/2 activity to test the effects of time and pressure. Comparisons
were performed using Bonferroni's test. Statistical significance was
accepted for values of P<.05.
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Results |
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Time Course of ERK1/2 Activation by Pressure In Vitro
As revealed in in-gel assays (Fig 1A
), increasing intraluminal pressure to
hypertensive levels (150 mm Hg) produced a rapid activation of
both ERK1 and ERK2, followed by a moderate decrease at 20 minutes and a
return to values close to baseline at 2 hours. Interestingly, after 24
hours of pressurizing at 150 mm Hg, ERK1/2 activity was again
increased and had a magnitude similar to that observed in the early
phase. In comparison, ERK1/2 activity in vessels maintained 24 hours at
10 mm Hg remained at baseline.
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Phosphorylation of ERK2 by MAP kinase kinase decreases
its electrophoretic mobility, thus allowing assessment of ERK2
phosphorylation state by Western blot.25,26
Using an anti-ERK1 antibody, we visualized changes in the proportion
between phosphorylated and
unphosphorylated ERK2 in the course of our experiments
(Fig 1B
). The appearance of the phospho-ERK2 band strictly correlated
with ERK1/2 activation, thus illustrating a direct relationship between
ERK1/2 phosphorylation and its enzymatic activity.
Quantification of in-gel assay results (Fig 1C) demonstrates that the
acute increase in intraluminal pressure to 150 mm Hg stimulated
ERK1/2 by 217.5±8.4% (n=6, P<.001) at 5 minutes. ERK1/2
activity decreased thereafter to 155.8±2.8% (n=6, P<.001)
at 20 minutes and reached 120.7±5.1% (n=8, P<.01) of
basal level by 2 hours. Sustained ERK1/2 activation observed after 24
hours of stimulation (241.8±14.7%, n=8, P<.001) was as
prominent as that noted at 5 minutes. The extent of pressure-induced
ERK1/2 activation was comparable to that obtained in freshly isolated
relaxed aortic segments exposed for 20 minutes to 10-6
mol/L PDBu (284.2±8.4%, n=3). Hence, early (5-minute) and late
(24-hour) ERK1/2 activations were separated by a period of lower,
almost baseline, activity at 2 hours, clearly underlining the biphasic
nature of ERK1/2 activation by pressure.
ERK1/2 Activation Is Pressure Dependent
To characterize long-term ERK1/2 activation in response to applied
intraluminal pressure, we analyzed vessel segments pressurized
in organ culture for 24 hours. ERK1/2 activity was moderately increased
by fixing intraluminal pressure at 50 mm Hg (111.0±4.6, n=8,
P<.05) or 80 mm Hg (122.6±5.5%, n=8,
P<.05) (Fig 2A and 2C)
compared with pressure maintained at 10 mm Hg. A further increase
in pressure to 150 mm Hg led to a dramatic increase in both ERK1
and ERK2 activity to 288±19.5% (P<.001, n=8) of that in
vessels maintained at 10 mm Hg during the course of experiment.
It is noteworthy that the extent of ERK1/2 activation induced by
pressurizing vessels at 150 mm Hg for 1 or 3 days was similar
(Fig 2A). A shift of electrophoretic mobility of ERK2 revealed on
Western blot with anti-ERK1 antibody (Fig 2B) reflects changes in ERK2
phosphorylation state and thus confirms measurements of
ERK1/2 activity obtained using the in-gel assay.
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Activation of Tyrosine Phosphorylation by
Pressure
Activation of tyrosine kinases in response to a variety of
extracellular stimuli occurs in the first minutes of cell
stimulation.27,28 Since ERK1/2 has been recognized among
the major targets for tyrosine phosphorylation, which
is essential for ERK1/2 activation,29 tyrosine
phosphorylation was studied in vessel segments
pressurized in vitro.
Total tyrosine phosphorylation in pressurized vessel
segments was assessed using Western blot with a phosphotyrosine
antibody. As outlined in "Materials and Methods," freshly isolated
segments were mounted on the pressurization and perfusion system and
equilibrated before the experiment in an incubator for 1.5 hours at
70 mm Hg. This experimental condition served as a control. Only
weak phosphotyrosine immunoreactivity was observed in these segments.
However, 5 minutes after increasing the intraluminal pressure from 70
to 150 mm Hg, a rapid activation of tyrosine
phosphorylation of protein bands with apparent
molecular masses ranging from 115 to 140 kD (Fig 3) as well as bands with other molecular
masses (180, 70, and 56 kD) was observed. Signals in the 42-and 44-kD
regions, presumably ERK1/2, were relatively weak compared with the
strong tyrosine phosphorylation of the bands in the
115- to 140-kD region. This probably reflects the fact that several
proteins are present in the 115- to 140-kD region, whereas there is
only ERK in the 42- and 44-kD bands. Indeed, 
90% of phosphotyrosine
immunoreactivity was detected in the 115- to 140-kD protein region. As
Fig 4 demonstrates, anti-phosphotyrosine
staining in segments during the first 2 hours of pressurizing was
rather uniform in the 115- to 140-kD region. Indeed, in contrast to the
biphasic ERK1/2 activation by pressure (Fig 1), activation of tyrosine
phosphorylation did not reveal "acute" and
"long-term" peaks but instead remained uniformly elevated between 5
minutes and 24 hours, with no reduction in tyrosine
phosphorylation at 2 hours (Fig 4A and 4B). However,
after 24 hours of pressurization at 150 mm Hg, we observed a
preferential accumulation of tyrosine phosphorylation
in the 125- to 140-kD region compared with the relatively uniform
tyrosine phosphorylation of proteins in the 115- to
125-kD and 125- to 140-kD regions at earlier time points (Fig 4C).
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The dependence of tyrosine phosphorylation on the
magnitude of applied pressure was investigated in greater detail in
1-day experiments. Total tyrosine phosphorylation,
including one major protein band with an apparent molecular mass of 120
to 130 kD, increased when applied intraluminal pressure was increased
(Fig 5). These results agree with a
pressure-dependent increase in ERK1/2 activity, as shown above.
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Effects of PKC or Tyrosine Kinase Inhibitors on ERK1/2
Activation in Pressurized Vessels
To verify whether PKC was involved in pressure-induced activation
of ERK1/2 in the vessels, aortic segments were cultured 24 hours at
150 mm Hg in the presence of PKC inhibitor
staurosporine or B-I. B-I is a highly specific
inhibitor of PKC, whereas staurosporine at high
concentrations has been shown to inhibit other kinases, such as
cGMP-dependent protein kinase30 and p34cdc2.31
Both inhibitors were used at concentrations capable of
inhibiting the contraction of freshly isolated vessel rings induced by
10 µmol/L PDBu (data not shown). Neither
staurosporine nor B-I suppressed pressure-induced ERK1/2
stimulation (Fig 6A and 6B), although
these inhibitors blocked PDBu-induced ERK1/2 activation in
vessel rings (data not shown). Interestingly, staurosporine
(10 µmol/L) stimulated the activation of a lower
molecular weight protein, probably p38 mitogen-activated
protein kinase (Fig 6A). This band was excluded from quantification of
ERK1/2. Both PKC inhibitors were also without effect in
short-term pressurizing experiments (data not shown).
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Because transmural pressure increased protein tyrosine
phosphorylation, we examined the effect of tyrosine
kinase inhibitors on ERK1/2 activation by transmural
pressure. At the concentrations used, these inhibitors were
found to block PDGF-induced ERK1/2 activation in aortic rings (data not
shown). Nonetheless, in acute or long-term experiments, tyrphostin A48
(50 µmol/L) or genistein (50 µmol/L) had no
effect on ERK1/2 activity. However, herbimycin A (500 nmol/L)
reduced pressure-induced ERK1/2 activation (150 mm Hg) by 92±8%
at 5 minutes (data not shown) and by 63±7% at 24 hours (Fig 7A and 7C). ERK
phosphorylation was also reduced at these early (not
shown) and late time points (Fig 7B).
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Discussion |
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Elevated blood pressure is associated with vascular remodeling, which is characterized by increased wall thickness, VSMC hypertrophy and/or hyperplasia, accumulation of extracellular matrix, and changes in vascular ultrastructure.1,2,32–34 This phenomenon is believed to involve mechanical effects of increased blood pressure, including stretch of vascular cells.4 However, in vivo experimental models of hypertension are too complex to allow separation of mechanical and neurohumoral components affecting the vessel wall. Using an organ culture model of pressurized rabbit aorta, we were able to evaluate the effects of increased intraluminal pressure on ERK1/2 activity. This is a key protein of the MAP kinase signaling cascade capable of controlling gene expression and cellular proliferation.18,35 In VSMCs, ERK1/2 may be activated by numerous stimuli, including growth factors and cytokines,36 vasoconstrictors,16,26,27 cellular contacts with matrix,37,38 and single mechanical stretch of aortic segments.15 However, the duration of MAP kinase activation is critical for MAP kinase effects on gene expression. For instance, the mitogenic effect of growth factors on cultured VSMCs correlates with the duration of ERK1/2 activation, but not with the extent of ERK1/2 activation, in the early phase.39 Therefore, since medial VSMCs are constantly under the influence of blood pressure, stretch may be a good candidate for regulation of gene expression via the ERK1/2 signaling cascade. In the present work, we found that aortic ERK1/2 was activated by elevated transmural pressure during prolonged periods, ie, up to 3 days in organ culture. As shown in in vivo experiments,21 acute hypertension led to transient ERK1/2 activation, which returned to the basal level 30 minutes thereafter. However, those authors did not explore longer periods of experimental hypertension to evaluate the possible reactivation of ERK1/2. We also observed a pronounced ERK1/2 activation 5 minutes after pressurizing the vessels at 150 mm Hg, which significantly decreased within the next 20 minutes. Others have shown that this primary peak correlates with the transient activation of phosphoinositide turnover, activation of PKC, and increased concentrations of intracellular Ca2+ 4,8 in response to a single or short-term stretch. Comparison of ERK1/2 activation and tyrosine phosphorylation profiles during short-term vessel pressurizing indicates that the early increase in ERK1/2 activity is accompanied by preferential uniform tyrosine phosphorylation of proteins in the 115- to 140-kD range.
More important, we observed a second phase of ERK1/2 activation in segments maintained at 150 mm Hg for 24 hours. In contrast to short-term experiments, segments pressurized for 24 hours were characterized by preferential increased tyrosine phosphorylation of protein bands in the 125- to 140-kD range. These results suggest that acute stimulation of the vessel segments by pressure may trigger several signaling pathways, resulting ultimately in activation of tyrosine kinases and ERK1/2. During longer pressurization periods, a different selection of signal-transducing pathways may be implicated. Thus, our data suggest the possibility of distinct signaling pathways recruited by short- and long-term mechanical stimulation.
Interestingly, ERK1/2 was activated moderately in the range of subphysiological values of pressure (10 or 50 mm Hg) or in normotensive conditions (80 mm Hg). In contrast, reproduction of the hypertensive state (150 mm Hg) was accompanied by sustained and dramatic (2.4-fold) ERK1/2 activation. We have previously shown that pressurizing the vessels in culture at 150 mm Hg stimulates protein synthesis and induces the expression of fibronectin by medial VSMCs without an increase in DNA synthesis.22,40 Thus, fibronectin expression and protein synthesis in our model of hypertensive vessel may be the consequences of sustained ERK1/2 activation and could reflect remodeling processes taking place in response to increased intraluminal pressure.
A role for PKC in stretch-mediated signaling in cardiomyocytes has been previously described.8 PKC also triggers tyrosine phosphorylation and ERK1/2 activation on phenylephrine stimulation in the ferret aorta.41 To test whether the observed sustained pressure-induced ERK1/2 activation was mediated by PKC, we incubated pressurized segments during 24 hours in the presence of the PKC inhibitor staurosporine or B-I. Neither of them abolished ERK1/2 activity. Moreover, vessels treated with a high concentration (1 µmol/L) of staurosporine caused the appearance of one more MBP-phosphorylating protein kinase with an apparent molecular mass of 38 kD, as revealed by in-gel assay. This kinase activity may be attributed to the p38/reactivating kinase group of stress-activated MAP kinases.42,43 This group of kinases is poorly activated by mitogens but potently activated by stressor agents, such as ischemia/reperfusion,44 heat shock, osmotic shock, or metabolic shock.45,46 This effect confirms that staurosporine penetrated the vessel segments and may indicate that high concentrations of the inhibitor imposed stress onto the cells of vessels cultured for 1 day. However, even at high concentrations, staurosporine was unable to inhibit ERK1/2 activation by pressure, despite the fact that both staurosporine and B-I slightly reduced total tyrosine phosphorylation in pressurized vessels. Thus, the present results suggest that acute and long-term activations of ERK1/2 in our model are PKC independent. This agrees with previous experiments showing that certain MAP kinase signaling pathways are PKC independent.47 Hence, the present results suggest that acute and long-term activations of ERK1/2 in our model are PKC independent, although a role for atypical PKC isoforms, which may be insensitive even to staurosporine or B-I, cannot be excluded.
On the other hand, possible effects of tyrosine kinase inhibitors were also investigated, since tyrosine phosphorylation of several proteins (especially those in the 115- to 140-kD range, but also those at 42 and 44 kD) was observed in parallel with pressure-induced ERK1/2 activation. Although ERK1/2 activation was not affected by the tyrosine kinase inhibitors tyrphostin A48 or genistein, it was partially (24 hours) or completely (5 minutes) inhibited by herbimycin A, an Src family kinase inhibitor.48 Our finding is in agreement with recent studies reporting that pp60c-Src is indeed activated by mechanical forces, such as shear stress in endothelial cells49 and strain in lung cells.50 The fact that herbimycin A was not as efficient at blocking long-term ERK1/2 activation compared with acute activation suggests that more than one signal transduction pathway may be involved in the late-phase activation of ERK1/2 by pressure.
Increased intraluminal pressure in cultured vessels activates the renin-angiotensin system in medial VSMCs.40 Furthermore, increased levels of Ang II in the media induces expression of fibronectin by VSMCs.51 Pressure-induced Ang II–dependent expression of fibronectin may be attenuated by the Ang II receptor antagonist losartan or the angiotensin-converting enzyme inhibitor lisinopril.40 To verify whether sustained pressure-induced ERK1/2 activation was also due to increased Ang II levels, we pressurized vessel segments in the presence of losartan (10 µmol/L). The Ang II receptor antagonist had no effect on pressure-dependent ERK1/2 activity (data not shown). Therefore, ERK1/2 activation in response to increased intraluminal pressure does not seem to be mediated by Ang II. These data do not rule out the possibility of ERK1/2 stimulation of angiotensin synthesis and subsequent fibronectin synthesis.
The presence of a major tyrosine-phosphorylated protein band in the 120- to 130-kD region could be due to FAK phosphorylation, since this protein has a molecular mass of 125 kD. Additional studies are required to confirm the identity of FAK as this band. Hamasaki et al52 recently showed that stretching cultured mesangial cells stimulates tyrosine phosphorylation of FAK. Since FAK is a major component of focal adhesion, activation of FAK by cyclic stretching52 or by pressurizing cultured vessels would support the hypothesis that focal adhesion may be an additional site where mechanical stress is translated into biochemical signals.
In summary, in the present work we investigated pressure-induced activation of ERK1/2 in an organ culture model of rabbit aorta. ERK1/2 activation was both biphasic and pressure dependent. It was also accompanied by activation of tyrosine kinases, one of which is possibly FAK. Pressure-induced ERK1/2 activation was PKC independent and was not triggered by Ang II, but it depended on a herbimycin A–sensitive tyrosine kinase. Significant and sustained ERK1/2 activation in vessel segments pressurized at 150 mm Hg in vitro correlates with stimulation of total protein and fibronectin synthesis by VSMCs reported previously.22,40 Thus, ERK1/2 may be involved in vascular remodeling triggered by elevated blood pressure.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Drs Birukov and Lehoux are recipients of fellowships from the Institut National de la Santé et de la Recherche Médicale (INSERM Poste vert and INSERM/Fonds de la Recherche en Santé du Québec, respectively). Support of Dr Birukov from Russian Fundamental Research Foundation grants 96-04-49274 and 96-04-49106 is also gratefully acknowledged.
Received May 14, 1997; accepted September 8, 1997.
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