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in vascular smooth muscle cells by mechanical stress
a Institute for Biomedical Aging Research, Austrian Academy of Sciences, University of Innsbruck Medical School, Innsbruck, Austria
b Institute for General and Experimental Pathology, University of Innsbruck Medical School, Innsbruck, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, an activated state. When GRB2, an adapter protein, was immunoprecipitated from treated VSMCs followed by Western blot analysis with anti-phosphotyrosine, -PDGFR, and -GRB2 antibodies, respectively, phosphotyrosine positive staining was observed on PDGFR bands of the same blot in stretch-stressed VSMCs, supporting the mechanical stress-induced activation of PDGFR. Conditioned medium from stretch-stressed VSMCs did not result in PDGFR phosphorylation, and antibodies binding to all forms of PDGFs did not block stress-induced PDGFR activation. Thus, mechanical stresses may directly perturb the cell surface or alter receptor conformation, thereby initiating signaling pathways normally used by growth factors.—Hu, Y., Böck, G., Wick, G., Xu, Q. Activation of PDGF receptor in vascular smooth muscle cells by mechanical stress. FASEB J. 12, 1135–1142 (1998)
Key Words: phosphorylation • MAP kinases • shear stress • AP-1 binding • platelet-derived growth factor receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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binds all forms of PDGFs, whereas receptor ß binds PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA (2, 3). The bind~ing of the ligand dimer induces dimerization of the platelet-derived growth factor receptors (PDGFRs), leading to their activation via autophosphorylation of tyrosine residues in the PDGFR kinase domain (4, 5). This triggers phosphorylation events involving sequential activation of mitogen-activated protein kinase (MAPK) cascades, which play a pivotal role in cell proliferation and differentiation (6). The passage of blood through the vascular system generates hemodynamic forces. Fluid flow across the cell surface results in shear stress, whereas tensile stress, which produces elongational stretch, is caused by circumferential deformations resulting from transmural pressure gradients and vascular smooth muscle tone (7). In vivo, factors ranging from physical exertion to psychological stress lead to a transient rise in blood pressure (8–10); if the factors are persistent and chronic, the arteriole walls gradually thicken, resulting in hypertension (8–12). Large arteries such as the aorta and coronary and carotid arteries undergo adaptation or remodeling in response to elevated blood pressure, showing medial hypertrophy and/or intimal hyperplasia of the arterial wall (13). There is evidence that mechanical stress initiates intracellular signaling (14–16) and stimulates the synthesis and/or secretion of various bioactive molecules including prostacyclin, nitric oxide synthase, PDGF, fibroblast growth factor (FGF), and transcription factor c-Fos/c-Jun (17). How vascular smooth muscle cells (VSMCs) sense mechanical perturbations remains to be elucidated.
We have shown previously that two distinct classes of MAPKs, the extracellular signal-regulated kinases (ERK) and the c-Jun N-terminal protein kinases (JNK), are transiently activated in rat arteries (aorta, carotid, and femoral arteries) in response to an acute elevation in blood pressure induced by either restraint (psychological stress) or administration of hypertensive agents (18). Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activator protein 1 (AP-1) DNA binding activity. Activation of ERK and JNK could contribute to smooth muscle cell hypertrophy/hyperplasia during arterial remodeling due to frequent and/or persistent elevations in blood pressure. However, we cannot distinguish the effects of hemodynamic stress or hormone/cytokine on this kinase activation in vivo. Nor is it known which molecules are responsible for initiation of MAPK activation in the arterial wall. In the present study, VSMCs were cultivated on a flexible membrane and subjected to mechanical stress, resulting in PDGFR-MAPK activation in VSMCs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cyclic strain stress
VSMCs were plated on silicone elastomer-bottomed culture dishes (Flexcell, Mckeesport, Pa.). Cells achieving 90% confluence were subjected to mechanical stress with the cyclic stress unit, a modification of the unit initially described by Banes et al. (21) consisting of a controlled vacuum unit and a baseplate to hold the culture dishes. Vacuum (15 to 20 kPa) was repetitively applied to the elastomer-bottomed dishes via the baseplate (Flexcell), which was placed in a humidified incubator with 5% CO2 at 37°C. Cyclic deformation (60 or 90 cycles/min) and 5–30% prolongation of elastomer bottomed dishes were used, comparable to previous work.
PDGF treatment
Subconfluent VSMCs were serum-starved for 3 days, and PDGF-AB (Sigma Chemical Co., St. Louis, Mo.) was added to the culture for various periods of time. Cells were harvested for PDGFR immunoprecipitation and Western blot analysis.
Shear stress treatment
The cone-and-plate apparatus was used to generate defined shear stresses, as described by Resnick et al. (22), with a modification. Briefly, the apparatus consists of a stainless steel cone rotating over a base plastic plate (100 mm in diameter; Costar), all maintained in a 5% CO2/95% air humidified atmosphere, thermostatically regulated at 37°C. Subconfluent VSMCs were serum-starved for 3 days and then exposed to laminar shear stress. Fluid mechanical parameters (medium viscosity, cone angle, and rotational speed) were adjusted to subject VSMCs to a uniform laminal shear stress of 5 dynes/cm2 (1 dyne = 100 mN) for various periods of time.
Protein extractions
For PDGFR isolation and MAPK assays, treated or untreated VSMCs were washed with cold (4°C) phosphate-buffered saline and harvested on ice in buffer A containing 20 mM Hepes (pH 7.4), 50 mM ß-glycerophosphate, 2 mM EGTA, 1 mM DTT, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 1 µg/ml leupeptin, 400 µM PMSF, and 1 µg/ml aprotinin. The suspension was incubated on ice for 15 min and centrifuged at 13,000 rpm for 30 min. The supernatant was harvested and protein concentration was measured with Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, Calif.).
Immunoprecipitation and Western blot analysis
For immunoprecipitation of PDGFR, GRB2, and ERK2, 0.5 ml of the supernatant containing 1 mg proteins was incubated with 10 µg of antibodies against PDGFR or GRB2 or with 1 µg of antibody against ERK2 (Santa Cruz Biochemicals, Santa Cruz, Calif.) for 3 h at 4°C with rotation. Subsequently, 80 µl of protein A-Sepharose 4B suspension (Sigma) was added and rotating continued for 1 h at 4°C. The immunocomplexes were precipitated by centrifugation and washed twice with buffers A, B (500 mM LiCl, 100 mM Tris, 1 mM DDT, 0.1% Triton X-100; pH7.6), and C (20 mM Mops, 2 mM EGTA, 10 mM MgCl2, 1 mM DDT, 0.1% Triton X-100; pH7.2), respectively, as described for the kinase assay (23, 24). The proteins were then separated by electrophoresis through 6% or 10% sodium dodecyl sulfate (SDS) -polyacrylamide gels and transferred to an immobilon-p transfer membrane. The membranes were processed with sequential antibodies against phosphotyrosine (4G10; Upstate Biotech. Inc., N.Y.), PDGFR, and GRB2, and the blot was stripped at 65°C for 30 min in buffer C before staining for another antibody. Specific antigen–antibody complexes were detected with the ECL Western Blot Detection Kit (Amersham). Photograms of the films were obtained in the linear range of detection and were quantified for the levels of specific band intensity by scanning laser densitometry (PowerLook II, UMAX Data System Inc., Hsinchu, Taiwan) of photograms.
Kinase assays
ERK2 activities in the immunocomplexes were measured as described previously (25–27). Briefly, immunocomplexes were incubated with 35 µl of buffer C supplemented with myelin basic protein (MBP; 6 µg; Sigma), 
-32P-ATP (15 µCi), MgCl2 (50 mM), and ATP (30 µM) for 20 min at 37°C, with vortexing every 3 min. To stop the reaction, 15 µl of 4x Laemmli buffer was added and the mixture was boiled for 5 min. Proteins in the kinase reaction were resolved by SDS-PAGE (polyacrylamide gel electrophoresis) (15% gel) and subjected to autoradiography.
Gel mobility shift assays
For nuclear protein preparation, the procedure was similar to that described by Schreiber (28) with slight modification. The treated cells were washed and harvested with cold TBS (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH7.4). The cell pellet (2–3x106) was incubated with 400 µl of cold buffer (10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DDT, 0.5 mM PMSF, pH7.9) on ice for 15 min after pipetting. Twenty-five microliters (10%) Nonidet P-40 was added, vortexed for 10 s, and centrifuged for 30 s at 13,000 rpm. The supernatant was used as cytosol proteins. The nuclear pellet was incubated with 50 µl of cold buffer (20 mM Hepes, 0.4 M NaCl, 20% glycerol, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 400 µM PMSF, 1µg/ml leupeptin, and 1µg/ml aprotinin, pH7.9) at 4°C for 15 min on a shaking platform with vigorous rocking. The suspension was centrifuged for 5 min at 4°C at 13,000 rpm. The supernatant was collected as nuclear proteins and protein concentration was determined with Bio-Rad assay.
The procedure was similar to that described previously (29, 30). In short, 5 µg of nuclear protein extracts was incubated with 0.5 ng of an oligonucleotide containing the AP-1 binding sequence (5'-CGCTTGATGACTCAGCCGGAA-3') labeled with [-32P]ATP. For the competition experiment, NF-
B oligonucleotide (5'-AGTTGAGGGGACTTT CCCAGGC-3') was also used. Reaction buffer contained 10 mM Tris, (pH 7.5) 1 mM DTT, 1 mM EDTA, 50 mM NaCl, 5% glycerol, and 1 µg poly(dIdC) as a nonspecific competitor. Samples were electrophoresed through a 4% polyacrylamide gel and exposed to autoradiographic film. Supershift assays were performed using antibodies against c-Fos (Santa Cruz Biochemicals). The antibodies were added to samples after the initial binding reactions between protein extracts, and oligonucleotides were allowed to take place.
Statistical analysis
Analysis of variance was performed when more than two groups were compared. A paired Student's t test was used to assess differences between two groups. A P value of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were examined by immunoprecipitation with a specific antibody against PDGFR from VSMCs treated with stretch stress or PDGF-AB and subsequent Western blot analysis with anti-phosphotyrosine antibodies. Stretch stress-stimulated PDGFR phosphorylation was observed as early as 4 min (
Fig. 1A).
Maximal phosphorylation was achieved 8 min after stretch stress (10% elongation) and returned to baseline by 60 min. As expected, exposure of VSMCs to PDGF-AB (100 ng/ml) for various periods of time stimulated PDGFR phosphorylation ( Fig. 1A) and was maintained at high levels for 4 to 20 min. Figure 1B summarizes data of PDGFR phosphorylation in percentage of PDGFR proteins as determined by quantification of optical densities from ECL photograms of three experiments. Thus, exposure of VSMCs to mechanical stress produced a three- to fivefold increase in PDGFR phosphorylation.
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To further study the effect of mechanical stress on PDGFR phosphorylation, VSMCs were stretched for prolongations of 5, 10, 20, and 30% of original size, respectively, or exposed to PDGF-AB for 8 min, and PDGFR phosphorylation in protein extracts was determined. Likewise, PDGFR phosphorylation was observed in magnitudes of stretch stress of 10–30%, but not 5% elongation ( Fig. 2),
indicating that a certain level of mechanical force is needed for PDGFR phosphorylation. In parallel studies, PDGF-AB-stimulated receptor phosphorylation was seen in concentrations of 10 to 100 ng/ml ( Fig. 2).
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Phosphotyrosine residues within the C-terminal part of the PDGFR constitute binding sites for several adapter molecules implicated in signal transduc~tion via their conserved SH2 domains. GRB2, an adapter protein, was immunoprecipitated from treated VSMCs with a specific antibody against GRB2, followed by Western blot analysis with anti-phosphotyrosine, -PDGFR, and -GRB2 antibodies, respectively. As shown in Fig. 3,
no significant difference in amounts of GRB2 and PDGFR proteins between treated and untreated cells was found. Phosphotyrosine positive staining was observed on PDGFR bands of the same blot in stretch-stressed VSMCs, supporting the mechanical stress-induced activation of PDGFR. A similar amount of PDGFR proteins was seen in either treated or untreated VSMCs, although PDGFR was not significantly phosphorylated in untreated cells. It might be a cell type-specific phenomena, since GRB2 forms a complex with activated PDGFR in other types of cells (31).
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Suramin, acting by destabilizing or preventing the formation of the active receptor conformation, blocked PDGFR phosphorylation in stretch-stressed VSMCs ( Fig. 4A).
These findings suggest that the initial signal in stressed VSMCs is generated at the plasma membrane. There is evidence that PDGFs are synthesized and released into medium in response to mechanical stress (32). To verify whether the ligand is involved in PDGFR activation, conditioned medium and neutralizing antibodies to PDGFs were used to treat cells. The data shown in Fig 4A indicate that supernatant or conditioned medium from stretch-stressed VSMCs (for 8 min) did not result in PDGFR phosphorylation. Furthermore, antibodies binding to all forms of PDGFs did not block stress-induced PDGFR phosphorylation ( Fig. 4B). Therefore, the secretion of growth factors in any measurable amount is too slow (4 h after stress) to account for the fast responses measured here.
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It has been shown that two forms of PDGFR exist in mammalian cells: mature PDGFR (180 kDa) and immature (160 kDa) (33). Our results demonstrated that the major PDGFR band was of the mature form (about 180 kDa) and a weaker band represented the immature form (see Figs. 1, 2, and 4). The phosphorylated PDGFR observed in the figures is the mature form.
ERK2 activation
PDGFR phosphorylation induced by ligand binding rapidly activates MAPK in a variety of cells in vitro (34–36). As shown above, PDGFR were phosphorylated or activated in mechanically stressed VSMCs. It should be possible to detect activation of ERK2-MAPKs in such cells; to provide indirect evidence of PDGFR activation, protein extracts from stressed VSMCs were analyzed for ERK2 activity determined using MBP as a substrate. The effect of this treatment on kinase activity is shown in Fig. 5.
Shear stress resulted in rapid ERK2 activation, with maximum activity (>fivefold elevation over untreated control) achieved within 8–20 min of shear stimulation. In parallel, a marked ERK activation was observed in stretched VSMCs treated with either 60 or 90 cycles for 10 min ( Fig. 5B). Concomitantly, supernatant from stretch-stressed VSMCs did not lead to ERK activation, and suramin completely blocked mechanical stress-stimulated activation ( Fig. 5C). In agreement with our findings, Reusch et al. (37) likewise showed that cyclin strain stress resulted in ERK activation in adult rat VSMCs independent of extracellular matrix composition. Taken together, these results suggest that mechanical stress-activated MAPKs may be, at least in part, induced by the growth factor receptor activation.
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AP-1 binding activation
MAPK regulates AP-1 transcription factor activity. Members of the Fos and Jun protein family dimerize to form AP-1 transcription factor complexes that regulate the expression of other genes (38, 39). It has been demonstrated that mechanical stress led to AP-1 activation in endothelial cells (40, 41). To determine whether AP-1 binding activity also increased in mechanically stimulated VSMCs, gel mobility shift assays using an oligonucleotide containing an AP-1 binding site were performed. Figure 6A
shows a time course for AP-1 activation in response to shear stress. Increased AP-1 binding activity was evident within 30 min of shear stimulation and maximum DNA binding was achieved 3 h after stress. Figure 6B also shows the results of gel mobility shift assays performed in the presence of unlabeled-AP-1 or NF-B oligonucleotides or antibodies specific to c-Fos proteins. The stress-induced increase in binding activity was specific for the AP-1, since increased concentrations of unlabeled AP-1 element effectively competed for binding to the factor whereas the NF-B binding element did not. Addition of antibody to the binding reaction resulted in a shift of the binding complexes to a slow migrating species, indicating the presence of Fos proteins in the DNA binding complexes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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What is the molecular mechanism of receptor activation by physical force? The mechanical stress activation of the PDGFR involves extremely rapid and suramin-sensitive phosphorylation of the receptor at tyrosine residues. With regard to the involvement of ligands in the stress-induced activation of growth factor receptors, our data do not support ligand binding activation. Whereas antibodies specific to the three forms of PDGFs block the growth factor response, we were not able to block any of the stress-induced fast responses with these antibodies. The conditioned medium from stressed cells resulted in neither PDGFR nor MAPK activation. Liu et al. (45) have demonstrated no measurable changes of PDGF in the culture medium within 4 h of mechanical strain. These results suggest that the activation of receptors by mechanical force does not involve the ligands and that suramin acts by destabilizing or preventing formation of the active receptor conformation.
Without ligand binding, how is phosphorylation or activation of growth factor receptors induced by mechanical force? The following options can be considered: physical force as energy is absorbed by growth factor receptors, by nonprotein or protein components of the plasma membrane other than the receptors, or by molecules at some other site of the cell, followed by transfer of the signal to the plasma membrane. Shyy and Chien (46) hypothesized that integrins may function as mechanotransducers that transduce the mechanical stress into chemical signals. There is evidence that induction of integrin clustering triggers the accumulation of focal adhesion kinases, protein tyrosine kinases, GRB2 and MAP kinases (47). These kinases present in cytoplasm might in turn phosphorylate PDGFR within the cell via a cross-talking mechanism. On the other hand, the possibility that the receptors themselves are directly targeted by the force is not ruled out, and would require that the receptors investigated can change into an active conformation upon energy uptake. Our results demonstrate that the extremely rapid tyrosine phosphorylation in PDGFR occurs in response to stress and that the time course of the stress-induced MAPK activation, followed by increased AP-1 binding activity, is similar to that observed in growth factor-stimulated response. These data support the notion of direct activation of the receptor. Obviously, there are discrepancies that still need to be resolved. Irrespective of the exact primary site of physical energy absorption, it is interesting that changes of the nuclear programs of gene expression are initiated by mechanical stress through the plasma membrane as if nuclei were primed to exclusively understand outside-in signals.
All tissues in the body are subjected to physical forces originating either from tension, created by cells themselves, or from the environment (48). The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized (49). Physical stimuli must be sensed by cells and transmitted through intracellular signal transduction pathways to the nucleus, resulting in physiological response or pathological conditions. For instance, cardiac hypertrophy, arteriosclerosis (50), some types of arthritis, and cancers are believed to be related to a sustained mechanical overloading or stress. Our findings of physical force-induced PDGFR activation give rise to the additional question of whether mechanical stress results in the activation of other growth factors such as adrenoceptor in myocytes, epithelial growth factor receptors in epithelial cells, and FGF receptors in synovial cells. Further study should provide valuable information for theoretical analysis and clinical application.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: AP-1, activator protein 1; ERK, extracellular signal-regulated kinases; FGF, fibroblast growth factor; JNK, c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PDGF, platelet-derived growth factor(s); PDGFR, platelet-derived growth factor receptor(s); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide electrophoresis; VSMCs, vascular smooth muscle cells.
Received for publication November 17, 1997. Accepted for publication April 10, 1998.
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