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(Hypertension. 2001;37:955.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Src Tyrosine Kinases and Extracellular Signal–Regulated Kinase 1/2 Mitogen-Activated Protein Kinases Mediate Pressure-Induced C-Fos Expression in Cannulated Rat Mesenteric Small Arteries

Jos P. M. Wesselman; Anca D. Dobrian; Suzanne D. Schriver; Russell L. Prewitt

From the Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk.

Correspondence to J.P.M. Wesselman, PhD, Department of Pharmacology and Toxicology, University Maastricht, PO Box 616, 6200 MD Maastricht, Netherlands. E-mail j.wesselman{at}farmaco.unimaas.nl

	Abstract

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Chronic hypertension is associated with remodeling of small arteries. There is evidence that the high pressure itself may cause these structural changes, but the responsible mechanisms are not clearly defined. Previously we showed that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries was inhibited by genistein, a general tyrosine kinase inhibitor. The purpose of this study was to further unravel the underlying signal transduction mechanisms, and we particularly tested the involvement of src tyrosine kinases and extracellular signal–regulated kinase (ERK). Rat mesenteric small arteries were cannulated in a dual-vessel chamber. After a 60-minute equilibration period, the pressure in 1 artery was increased to 140 mm Hg, while the other artery remained at 90 mm Hg. Semiquantitative reverse transcriptase–polymerase chain reaction was used to determine c-fos expression, and Western blotting was used to examine levels of ERK phosphorylation. The involvement of src and ERK was tested with the inhibitors herbimycin A (1 µmol/L), PP1 (10 µmol/L), PP2 (10 µmol/L), and PD98059 (30 µmol/L). One-hour exposure to 140 mm Hg increased the c-fos/cyclophilin ratio 3.6-fold, from 0.29±0.07 to 1.06±0.25. All the tested inhibitors suppressed the pressure-induced increase of c-fos expression. A 5-minute exposure period to 140 mm Hg increased ERK phosphorylation, and this was abolished in the presence of PP1. The results suggest that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries may be mediated, at least in part, by src tyrosine kinases and ERK.

Key Words: arteries • remodeling • pressure • signal transduction • proto-oncogene proteins c-fos • src-family kinases • protein kinases

	Introduction

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The development of hypertension is associated with remodeling of small arteries (diameter, 100 to 500 µm). These structural changes include a reduced lumen diameter, an increased media/lumen ratio, and, depending on the kind of hypertension and the location of the vessel bed, an increased or unchanged medial cross-sectional area.^{1 2 3 4} This indicates the involvement of either inward hypertrophic growth or eutrophic remodeling.^{5 6} There is evidence that the high pressure itself may be responsible for this remodeling process. Bund et al⁷ showed that reduction of the femoral arterial pressure, by use of a partially constricting ligature, prevented structural changes of femoral resistance arteries in spontaneously hypertensive rats. Pressure may also be the mediator of the outward hypertrophic remodeling response in large arteries. Parker et al⁸ found that minoxidil, a K_ATP-channel opener, reversed both blood pressure and aortic hypertrophy in angiotensin II–induced hypertension.

The hypothesis that pressure activates mechanisms that may eventually lead to arterial remodeling has been investigated in cultured vascular smooth muscle cells. Mechanical stimulation, such as the application of pressure, stretch, or deformation, of cultured arterial smooth muscle cells has been shown to induce a growth response.^{9 10} Furthermore, it was found that this type of stimulation induced the activation of the extracellular signal–regulated kinase (ERK) and c-jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase cascades^{11 12 13 14} ; expression of proto-oncogenes, such as c-fos, c-myc, and Egr-1^{9 15 16 17} ; release of growth factors, such as platelet-derived growth factor (PDGF) A,¹⁰ insulin-like growth factor I,¹⁸ and fibroblast growth factor-2¹⁹ ; expression of connexin 43¹⁶ ; and synthesis of smooth muscle myosin,²⁰ collagen,²¹ and elastin.¹⁵ These results demonstrate that mechanical stimulation indeed activates mechanisms that promote remodeling or growth in these cells. However, several studies clearly show that the responses to mechanical stimulation are dependent on the plating medium and the specific adhesion molecules that are engaged.^{11 17 22} This suggests that the candidate signaling pathways, as found in cultured cells, should be confirmed in an actual intact artery with a natural cell phenotype and matrix.

Thus far, only a few studies that sought to determine the mechanisms by which pressure induces a growth or remodeling response have been performed on intact arteries. Xu et al²³ demonstrated that acute hypertension activated ERK and JNK MAP kinases and increased expression of c-fos and c-jun proto-oncogenes and AP-1 binding activity in rat large arteries in vivo. In our group, work from Parker et al⁸ and Dobrian et al²⁴ suggests that the aortic and femoral artery hypertrophy in angiotensin II–induced and 1-kidney, 1 clip hypertensive rat is mediated by pressure-induced expression of PDGF-A. Furthermore, Adam et al²⁵ showed that the application of mechanical load rapidly, within a minute, increased levels of ERK MAP kinase activity in isolated strips of porcine carotid artery. Birukov et al²⁶ found that pressurization of cannulated rabbit aorta in organ culture activated ERK. In small arteries, which show a different remodeling response, pressure may activate other signal transduction mechanisms. We previously showed that elevating intraluminal pressure from 90 to 140 mm Hg in cannulated rat mesenteric small arteries increased the expression of c-fos and c-myc.^{27 28} Recently we showed that this response did not depend on intracellular calcium, protein kinase C, or intact actin filaments but was inhibited by genistein, a tyrosine kinase inhibitor.²⁹

In the present study we sought to further characterize the mechanotransduction mechanism by which pressure induces proto-oncogene expression in intact small arteries. In particular, we tested, using specific inhibitors, whether pressure-induced c-fos expression in cannulated rat mesenteric small arteries required src-family tyrosine kinases. Src tyrosine kinases are associated with integrins at the focal adhesion sites. Here, the integrins connect the extracellular matrix with the intracellular cytoskeleton and may thus mediate mechanotransduction of the pressure stimulus.³⁰ Second, we examined the role of ERK MAP kinases, which, like c-fos, have been associated with proliferative responses to mechanical stimulation.³¹

	Methods

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Isolation Artery Experiments
All experimental procedures were approved by the institutional Animal Care and Use Committee. Male Wistar rats (weight, 263±7 g) were anesthetized with pentobarbital (60 mg/kg IP). After laparotomy and removal of the heart, the mesenteric arcade was dissected and placed in a cold bicarbonate-free physiological saline solution, with the following composition (in mmol/L): NaCl 141.8, KCl 4.69, MgSO₄ 1.59, EDTA 0.513, CaCl₂ 2.79, HEPES 10, KH₂PO₄ 1.18, glucose 5, at pH 7.4. First-generation mesenteric arteries were cleared from surrounding adipose and connective tissue and cannulated onto 2 glass micropipettes in a dual-vessel chamber (model CH/2/M, Living Systems Instrumentation). The pressure myograph was transferred to the stage of an upright microscope. The image of the transilluminated vessel was recorded with a video camera, and the internal and external diameters were measured with a video caliper (Texas A&M). The intraluminal pressure could be controlled by adjustment of the height of a fluid reservoir, which was connected to one of the cannulas.

For the c-fos experiments, we followed a previously described protocol for altering pressure.^{27 28 29} During a 1-hour equilibration period, the intraluminal pressure was raised in small steps of 15 mm Hg, from 30 to 90 mm Hg, at 37°C. After equilibration, the intraluminal pressure in 1 artery was increased to 140 mm Hg in 1 step to simulate an acute hypertensive situation, while the other artery was maintained at 90 mm Hg, the physiological pressure.³² These pressures were maintained for 1 hour. Subsequently, the arteries were removed from the cannulas, and c-fos expression was determined by means of reverse transcriptase–polymerase chain reaction (RT-PCR). For the experiments in which ERK MAP kinase phosphorylation was analyzed, the arteries were equilibrated at 90 mm Hg for a period of 1 hour at 37°C. Subsequently, 1 of the 2 vessels was subjected to an intraluminal pressure of 140 mm Hg for 1, 5, or 10 minutes, while the other artery remained at 90 mm Hg. Then the arteries were removed from the cannulas, and ERK phosphorylation was analyzed by Western blotting.

Experiments were always performed in a paired fashion; the 2 arteries that were compared were taken from the same rat. To further reduce the variability of the c-fos experiments, 2 isolated arteries were obtained by cutting 1 vessel into 2 segments of similar axial length (2 to 3 mm). This was not possible for the experiments in which levels of ERK MAP kinase phosphorylation were determined because more tissue was needed for the assay. All inhibitors were administered extraluminally in the tissue bath during the 1-hour incubation period.

RNA Isolation and RT-PCR
Expression of c-fos was determined by RT-PCR. After the pressure protocol, the arteries were quickly removed from the cannulas and stored in RNAlater (Ambion). The vessels were carefully ground in cold guanidinium isothiocyanate and 2-mercapto-ethanol with the use of glass tissue grinders. RNA was isolated and purified on a GlassMax RNA Microisolation Spin Cartridge System (GIBCO BRL). The quantity (usually between 150 and 1000 ng per artery) and purity of the RNA were determined by spectrophotometry using a Genequant device (Pharmacia). The RT was performed with the use of a Reverse Transcription System (Promega). Care was taken to perform the RT reaction with the same quantity of RNA (100 ng). One fourth of the RT volume (5 µL cDNA template) was amplified with primers (GIBCO BRL) for both c-fos and cyclophilin. Cyclophilin was used as the housekeeping gene for semiquantitative analysis of c-fos expression because it has been demonstrated to be abundantly expressed in vascular smooth muscle and to be refractory to many stimuli, including mechanical loading.³³ For the PCR, a Taqbead Hot Start Polymerase (Promega) was used. The cDNA and 1.25 U of Taq polymerase were added to 45 µL of master mix. The samples underwent 30 cycles of denaturation (94°C), annealing (52°C), and extension (72°C). The PCR product was electrophoresed on an 8% polyacrylamide gel. The bands were visualized with the use of the EagleEye II Still Video System (Stratagene) and quantified by means of SigmaGel software (SPSS Inc). For amplification of c-fos cDNA, the sense primer was 5'-GAT-GTT-CTC-GGG-TTT-CAA-CGC-G-3', and the antisense primer was 5'-TGC-AGC-CAT-CTT-ATT-CCT-TTC-CC-3', which gave a 451-bp DNA product. The c-fos primers were chosen such that they span 3 introns. The primer sequences for cyclophilin were sense: 5'-GTC-GCG-TCT-GCT-TCG-AGC-TGT-TTG-C, and antisense: 5'-CCA-TGG-CTT-CCA-CAA-TGC-TCA-TGC-C-3', which gave a DNA band of 375 bp.

Western Blotting
ERK 1/2 MAP kinase phosphorylation was detected by Western blotting. After the pressure protocol, the arteries were quickly removed from the cannulas and immediately ground with glass tissue grinders in ice-cold buffer (50 µL) of the following composition: Tris-HCl 50 mmol/L (pH 7.4), NP-40 1%, Na-deoxycholate 0.25%, NaCl 150 mmol/L, EDTA 1 mmol/L, phenylmethylsulfonyl fluoride 1 mmol/L, aprotinin 1 µg/mL, leupeptin 1 µg/mL, pepstatin 1 µg/mL, Na₃VO₄ 1 mmol/L, NaF 1 mmol/L (final concentrations). The protein concentration was measured with the bicinchoninic acid protein assay. Equal amounts of protein (10 µg) were separated by electrophoresis on a 10% polyacrylamide gel and electroblotted on a PVDF transfer membrane blocked with Tris-buffered saline/Tween and NAP-Sureblocker (Geno Technology). Blots were incubated with a phosphorylation-specific primary antibody for ERK 1/2 MAP kinase (Santa Cruz Biochemicals), followed by a horseradish peroxidase–labeled secondary antibody (Promega). Antibody complexes were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). Prestained rainbow markers (Amersham) were used as molecular mass standards.

Chemicals
PP2 was obtained from Calbiochem-Novabiochem Corp, PD98059 from New England BioLabs Inc, and herbimycin A and PP1 from Biomol Research Laboratories, Inc. All other compounds were purchased from Sigma Chemical Co.

Data Analysis
Results are presented as mean±SEM. Statistical significance was tested by ANOVA followed by Bonferroni multiple comparison test. The null hypothesis was rejected at P<0.05, and n depicts the number of rats.

	Results

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Sixty pairs of rat first-generation mesenteric arteries were used in this study. Under control conditions, their mean internal diameter at 90 mm Hg, an approximation of the physiological pressure,³² was 362±11 µm (n=17). During equilibration, most arteries displayed a pressure response that corresponds with passive characteristics: an increase in diameter immediately after pressure elevation (especially between 30 and 75 mm Hg), which was followed by a slow and small further increase to reach a stable steady state diameter after 3 to 5 minutes. In approximately 35% of the control arteries, pressurization to either 90 or 140 mm Hg resulted in a moderate myogenic responsiveness, which was observed in earlier studies.^{28 34} However, this low level of smooth muscle tone, as well as the pharmacological inhibitors, did not significantly influence steady state diameters at 90 and 140 mm Hg. Moreover, in all experimental groups, diameters at 90 and 140 mm Hg were not significantly different, demonstrating that the pressure elevation to 140 mm Hg does not induce additional stretch of the individual smooth muscle cells. Furthermore, the circumferential wall stress of the arteries was always increased at 140 mm Hg compared with 90 mm Hg. Exposure to the higher pressure, 140 mm Hg, caused a >3-fold increase in c-fos expression (n=8), as determined by RT-PCR: the mean c-fos/cyclophilin ratio increased from 0.29±0.08 at 90 mm Hg to 1.06±0.25 at 140 mm Hg (Figures 1 and 2).

View larger version (29K): [in this window] [in a new window]   Figure 1. Example of a polyacrylamide gel that shows RT-PCR products of c-fos (451 bp) and cyclophilin (375 bp) from 2 small mesenteric arteries that have been exposed to 90 mm Hg (left) and 140 mm Hg (right). In this typical experiment the c-fos/cyclophilin ratios were 0.59 at 90 mm Hg and 1.11 at 140 mm Hg.

View larger version (17K): [in this window] [in a new window]   Figure 2. Summary of the effects of inhibitors of src tyrosine kinases and the ERK MAP kinase pathway on pressure-induced c-fos expression. A, Effects of pressure on c-fos/cyclophilin ratios under control conditions (n=8), in the presence of herbimycin A (1 µmol/L, n=10), PP1 (10 µmol/L, n=10), PP2 (10 µmol/L, n=10), and PD98059 (30 µmol/L, n=10). B, Same data displayed as difference between c-fos/cyclophilin ratio at 90 and 140 mm Hg. *P<0.01; 1-way ANOVA performed on all c-fos/cyclophilin ratios (arranged in 10 groups as depicted in A) produced a P-value of 0.0005, and additional Bonferroni multiple comparison (90 vs 140 mm Hg in all groups) only showed significance in the control group (P<0.01).

Effects of Inhibitors for Src Tyrosine Kinase and the ERK Cascade on C-Fos Expression
To determine the involvement of src tyrosine kinases in pressure-induced c-fos expression, we used the src inhibitors herbimycin A (1 µmol/L, n=10), PP1 (10 µmol/L, n=10), and PP2 (10 µmol/L, n=10). Figure 2 summarizes the results. Herbimycin A and PP1 did not influence c-fos expression at 90 mm Hg but suppressed the additional expression induced by pressure elevation; the c-fos/cyclophilin ratios at 90 and 140 mm Hg were not significantly different (Figure 2A). Unexpectedly, in the presence of PP2 basal c-fos levels appeared to be increased. However, like the other src inhibitors, it also blocked the pressure-induced rise of the c-fos/cyclophilin ratio. Thus, all the src tyrosine kinase inhibitors blocked the increase of c-fos expression that was induced by high pressure (Figure 2B). The specific inhibitor of MAP kinase/ERK kinase (MEK) PD98059 (30 µmol/L, n=10) was used to assess the involvement of the ERK 1/2 MAP kinase pathway in pressure-induced c-fos expression. Figure 2 demonstrates that, although basal c-fos levels were increased, high pressure did not influence the c-fos/cyclophilin ratio.

Pressure-Induced ERK MAP Kinase Phosphorylation
To further unravel the involvement of the ERK MAP kinase pathway, we investigated whether high pressure induces phosphorylation. By means of Western blotting, with a phosphorylation-specific primary antibody for ERK 1/2, we investigated whether exposure of cannulated mesenteric small arteries to high pressure evokes ERK phosphorylation. In 3 different sets of experiments, the arteries were subjected to high pressure for 1, 5, and 10 minutes (3 pairs of arteries for each period). One minute at 140 mm Hg changed ERK phosphorylation to 119%, 1042%, and 27%, 5 minutes at 140 mm Hg to 560%, 302%, and 166% (Figure 3A), and 10 minutes at 140 mm Hg to 157%, 40%, and 62%. These results show that a 1-minute, as well as a 10-minute, exposure to high pressure did not uniformly change ERK phosphorylation, whereas a 5-minute exposure to 140 mm Hg consistently increased ERK phosphorylation in all 3 experiments (Figure 3A). To investigate whether pressure-induced ERK phosphorylation depends on src tyrosine kinases, we tested whether PP1 (10 µmol/L, n=3) influenced the response to 5-minute exposure to 140 mm Hg. Figure 3B shows that levels of ERK phosphorylation were extremely low in 1 pair of arteries, both under control conditions and after 5 minutes at 140 mm Hg. For the 2 other pairs that were treated with PP1, phosphorylation was not detectable at either pressure (data not shown). Collectively, the results show that blockers of src tyrosine kinases and MEK inhibited pressure-dependent c-fos expression and that src blockade suppressed ERK-phosphorylation as well.

View larger version (42K): [in this window] [in a new window]   Figure 3. Effect of PP1 on pressure-induced ERK 1/2 MAP kinase phosphorylation. A, These blots show that, in 3 experiments, a 5-minute exposure to 140 mm Hg increased ERK phosphorylation compared with 90 mm Hg. B, In the presence of PP1 (10 µmol/L, n=3), ERK phosphorylation was extremely low at 90 mm Hg and after 5 minutes at 140 mm Hg as well. For the other 2 pairs, ERK phosphorylation at 90 and 140 mm Hg was not detectable.

	Discussion

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The main results of the present study are that, in intact cannulated rat mesenteric small arteries, the earlier reported pressure-induced increase of c-fos expression^{27 28 29} was suppressed by 3 different src tyrosine kinase inhibitors and by blockade of the ERK MAP kinase cascade. Furthermore, we found that high pressure activated ERK MAP kinases as well, and that this response was abolished by src blockade. These results suggest that activation of both src tyrosine kinases and ERK MAP kinases is required for the transduction of the pressure stimulus to c-fos expression and that src is upstream from ERK in the signaling pathway.

Protein tyrosine kinases consist of several subclasses, and some of those, eg, focal adhesion kinase or src, are associated with integrins. Integrins are transmembrane adhesion molecules that couple the extracellular matrix to the cytoskeleton at the focal adhesion sites, and evidence is accumulating that integrins are involved in the transduction of mechanical stimuli.^{30 31} Results from several studies suggest that interactions between integrins and specific matrix proteins may be critically important for the transduction of mechanical stimulation, which eventually leads to a mitogenic response. Wilson et al²² showed that mechanical strain increased DNA synthesis in vascular smooth muscle cells on collagen, fibronectin, or vitronectin but not on cells on elastin or laminin. Antibodies to both ß₃ and _vß5 integrins, but not antibodies to ß₁ integrins, inhibited strain-induced DNA synthesis. Furthermore, they found that RGD peptide blocked the strain-induced mitogenic response and expression and secretion of PDGF. Other studies demonstrated that, in neonatal rat vascular smooth muscle cells, strain-induced activation of ERK MAP kinases, expression of smooth muscle myosin, and expression of the immediate early genes Egr-1 and c-jun were dependent on the composition of the plating medium.^{11 17} These studies indicate that it is not indiscriminate stretch of the cells that initiates a response but rather that adhesion molecules are acting as specific receptors that determine the nature of the response. Which focal adhesion sites are formed and capable of participating in the signaling pathway depends on the surrounding matrix. In the present study we used the isolated small artery preparation to ensure physiologically relevant cell-cell and cell-matrix interactions.

The current results show that 3 different src family tyrosine kinase inhibitors, herbimycin A, PP1, and PP2, all suppressed the pressure-induced rise in c-fos expression. Because PP1 and PP2 are highly specific for src tyrosine kinases,³⁵ these findings suggest that activation of src is required for pressure-induced c-fos expression. Why baseline c-fos levels at 90 mm Hg were increased in the presence of PP2 is not clear. Hanke et al³⁵ showed that PP1 and PP2 are closely related pyrazolopyrimidines, but, although they are both extremely potent and selective src inhibitors, their inhibitory action on the different src family kinases shows some degree of variation. Nevertheless, the finding that PP2 blocked the pressure-induced increase in gene expression is consistent with the effects of the other src inhibitors.

The ERK MAP kinase pathway has been associated with vascular remodeling in response to hypertension. In vivo experiments showed rapid activation of ERK in rat aorta in response to acute hypertension.²³ Likewise, mechanical stimulation of large arteries in vitro or cultured vascular smooth muscle cells caused quick activation of ERK.^{12 13 25 26} Moreover, it is generally accepted that activation of ERK MAP kinases leads to proto-oncogene expression and eventually to a growth response.^{30 31 36} The present results show, for the first time, that high pressure rapidly phosphorylates ERK in intact small arteries. A 1-minute, as well as a 10-minute, exposure to high pressure did not uniformly change ERK phosphorylation, but a 5-minute exposure to 140 mm Hg consistently increased ERK phosphorylation in all 3 experiments (Figure 3A). This suggests that in intact small arteries ERK phosphorylation in response to high pressure may be biphasic in time, as was found by others in different experimental settings^{13 23 26} ; a 1-minute exposure may in some cases not be sufficient to start ERK phosphorylation, which appears to peak after 5 minutes, and after 10 minutes levels of ERK phosphorylation in some arteries may have returned to (or even below) baseline. The involvement of the ERK cascade in pressure-induced c-fos expression was tested by means of PD98059. For unknown reasons c-fos levels at 90 mm Hg were elevated compared with control at 90 mm Hg. One might speculate that other MAP kinases may have compensated for the inhibition of ERK, but obviously this needs to be tested. Nevertheless, in the presence of PD98059, high pressure did not increase c-fos expression. Thus, the results not only indicate that ERK is activated by pressure but also suggest that its activation is essential for the transmission of the pressure stimulus to gene expression. The finding that levels of ERK phosphorylation are hardly or not detectable in the presence of PP1 suggests that activation of src is required for both basal, pressure-independent, ERK phosphorylation and for the propagation of the pressure stimulus to subsequent ERK phosphorylation and c-fos expression. One could argue that PP1, even though it is supposed to be highly specific for src, blocked ERK phosphorylation directly. Although we cannot rule out this possibility, we are not aware of any evidence for such nonspecific PP1 activity. Therefore, we believe that these findings reflect the requirement of src activation for the propagation of the mitogenic transduction pathway. This view is consistent with results from earlier studies on other preparations. Davis et al⁹ showed that herbimycin A prevented the cyclic deformation-induced vascular smooth muscle growth. Birukov et al²⁶ found that herbimycin A inhibited pressure-induced ERK activation in cannulated rabbit aorta in culture.

This kind of mechanotransduction appears not to be restricted to vascular smooth muscle cells. Similar mechanisms have been described for the transduction of fluid shear stress on vascular endothelial cells³⁷ or the response of cardiac myocytes to mechanical stress.³⁸ Combining the present observations with results from other studies, we propose the following model for the mechanotransduction of pressure to c-fos expression in rat mesenteric small arteries (Figure 4). High pressure increases wall stress, which may act on the integrins at the focal adhesion sites. This may activate src tyrosine kinases, which are linked to the ß-subunit of the integrins, allowing association with the adaptor protein complex Shc-Grb2-Sos. This would promote GTP binding on ras, phosphorylation, and activation of raf (MAPKKK), MEK (MAPKK), and ERK (MAPK). ERK may finally stimulate c-fos expression through phosphorylation of the TCF transcription factor (Figure 4).

View larger version (25K): [in this window] [in a new window]   Figure 4. Possible mechanotransduction pathway by which pressure induces c-fos expression. High pressure increases circumferential wall stress, which may act on the focal adhesion sites, containing transmembrane integrins that link the extracellular matrix (ECM) with a complex of cytoskeletal proteins. This may activate tyrosine kinases, such as src and focal adhesion kinase (FAK), which are linked with the ß-integrin subunit, allowing association with the adaptor protein complex Shc-Grb2-Sos. This would promote GTP binding on ras, phosphorylation, and activation of raf (MAPKKK), MEK (MAPKK), and ERK (MAPK). ERK may finally stimulate c-fos expression through phosphorylation of the TCF transcription factor. SRF indicates serum response factor; SRE, serum response element; and IE gene, immediate early gene.

It is, however, quite likely that the possible scenario depicted in Figure 4 is incomplete. We do not exclude other factors that are activated by mechanical stimulation and may act in concert with the aforementioned reaction cascade. In cultured vascular smooth muscle cells, it was found that mechanical stress induced phosphorylation of the PDGF and the epidermal growth factor receptor. Hu et al³⁹ showed that antibodies to PDGF did not block this response, suggesting that mechanical stimulation may directly activate the PDGF receptor. Activation of the receptor leads to autophosphorylation of tyrosine residues within the receptor and downstream activation of src, Shc, Grb2, Sos, and ERK.³⁶ In a recent report, Iwasaki et al¹⁴ described a similar mechanism for the epidermal growth factor receptor. Waltenberger and coworkers⁴⁰ showed that PP1, apart from its src-inhibitory action, directly blocked PDGF receptor tyrosine kinase activity. Thus, we cannot exclude that the PP1 effects in the present study were caused by blockade of the PDGF receptor rather than src inhibition.

In summary, we confirmed the previously found pressure-induced increase in c-fos expression in intact cannulated rat mesenteric small arteries. Specific inhibition of src tyrosine kinases and the ERK MAP kinase cascade blocked this effect of pressure. In addition, elevated pressure increased ERK phosphorylation, which was inhibited by PP1. These results suggest that activation of src tyrosine kinases and the ERK pathway may mediate pressure-induced c-fos expression in intact rat mesenteric small arteries.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant HL 54810 and a Postdoctoral Fellowship from the Mid-Atlantic Region of the American Heart Association to Dr Wesselman.

Received April 18, 2000; first decision May 18, 2000; accepted August 31, 2000.

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