The migration of vascular smooth muscle cells (SMCs) and fibroblasts into the intima after vascular injury is a central process in vascular lesion formation. The elevation of transmural interstitial flow is also observed after damage to the vascular endothelium. We have previously shown that interstitial flow upregulates matrix metalloproteinase-1 (MMP-1) expression, which in turn promotes SMC and fibroblast migration in collagen I gels. In this study, we investigated further the mechanism of flow-induced MMP-1 expression. An ERK1/2 inhibitor PD-98059 completely abolished interstitial flow-induced SMC migration and MMP-1 expression. Interstitial flow promoted ERK1/2 phosphorylation, whereas PD-98059 abolished flow-induced activation. Silencing ERK1/2 completely abolished MMP-1 expression and SMC migration. In addition, interstitial flow increased the expression of activator protein-1 transcription factors (c-Jun and c-Fos), whereas PD-98059 attenuated flow-induced expression. Knocking down c-jun completely abolished flow-induced MMP-1 expression, whereas silencing c-fos did not affect MMP-1 expression. Taken together, our data indicate that interstitial flow induces MMP-1 expression and SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism and suggest that interstitial flow, ERK1/2 MAPK, c-Jun, and MMP-1 may play important roles in SMC migration and neointima formation after vascular injury.
- shear stress
- matrix metalloproteinase
- mitogen-activated protein kinase
- activator protein-1
- neointima formation
- smooth muscle cell
- extracellular signal-regulated kinase
vascular smooth muscle cell (SMC) and fibroblast migration and proliferation in the intima play a major role in neointima formation after vascular injury. It is well known that growth factors and inflammatory cytokines can induce SMC migration through three-dimensional (3-D) extracellular matrix (ECM) by stimulating the secretion of matrix metalloproteinases (MMPs), which are capable of digesting ECM (15, 25, 48). For example, the upregulated expression of MMP-1 in the early stages of atherosclerosis is associated with SMC migration (2, 3). Because the major collagen components in the vascular wall are collagen type I and type III and because MMP-1 is responsible for the initial cleavage of collagen I and III, MMP-1 is believed to play an essential role in vascular SMC migration (15).
Transmural interstitial flow driven by the transmural pressure differential is a physiological fluid movement through the vascular vessel interstitium that imposes fluid shear stress on parenchymal cells (41, 44). The biological role of this small flow (shear stress) has not been well recognized (39, 40, 45). However, during the early stages of vascular injury, the interstitial flow is elevated because of a loss of endothelial hydraulic resistance, and we hypothesized that this elevated flow could participate in vascular SMC and fibroblast migration and neointima formation (39). Furthermore, in hypertension, the transmural interstitial flow is also increased because of the elevated transmural differential pressure in the large arteries, which also can lead to neointima formation (20, 26). We have previously shown that interstitial flow can stimulate SMCs and fibroblasts to express MMP-1, which can in turn facilitate cell migration in collagen I gels (39). However, one of the remaining challenges is to determine the exact biomolecular signaling pathway (mechanism) of flow-induced MMP-1 expression.
Therefore, in this study, we investigated the underlying mechanism of interstitial flow-induced MMP-1 expression. The activation of mitogen-activated protein kinases (MAPKs) such as extracellular signal-related kinase-1 and -2 (ERK1/2), the expression of activator protein-1 (AP-1) transcription factors such as c-Jun and c-Fos, and the AP-1 DNA binding activity were examined. Gene silencing was also conducted to confirm the roles of ERK1/2 and AP-1 in the regulation of MMP-1 expression. The results suggest that interstitial flow induces MMP-1 expression and SMC migration via an ERK1/2-dependent AP-1 (c-Jun) activation mechanism.
MATERIALS AND METHODS
Collagen gel preparation and flow experiments.
Rat aorta SMCs were isolated from male Sprague-Dawley rats weighing 150 g (16). The procedure was approved by the City College/City University of New York Medical School Animal Care and Use Committee. As previously described (39), rat aortic SMCs (passages 3–5) were suspended in rat-tail collagen I (BD Science) gels (cell density, 2.5 × 105 cells/ml; and final gel concentration, 4 mg/ml), and pH was adjusted to 7.0 by mixing the appropriate amount of NaOH. For cell migration experiments, 200 μl of gel were loaded into each 12-well cell culture insert with 8-μm pores (BD Science). For RNA extraction and protein extraction experiments, 6-well cell culture inserts with 8-μm pores (BD Science) were used. To keep the same level of shear stress, the same gel thickness was maintained in both the 6- and 12-well experiments, thus 1 ml of gel was used for a 6-well insert. The gels were incubated for 24 h to allow cell spreading. Gels were then subjected to interstitial flow driven by a 1-cmH2O pressure drop (shear stress was ∼0.05 dyn/cm2) for various time periods according to the specific experimental design. This shear stress level elicited the maximum enhancement of migration in our previous study (39). For MAPK inhibition experiments, after 24 h of cell spreading in the collagen gel, the cells were treated with p38 MAPK inhibitor InSolution SB-203580 (Calbiochem) or ERK1/2 inhibitor InSolution PD-98059 (Calbiochem) for 1 h followed by various time periods of interstitial flow with or without inhibitors in the flow media.
Western blot analysis.
Collagen gels were washed once with ice-cold PBS, and 2× lysis buffer was then added immediately to the gels followed by a sonication for 30 s on ice. The 2× lysis buffer was composed of 2× radioimmunoprecipitation assay buffer, containing 300 mM NaCl, 2% Nonidet P-40, 100 mM Tris, 0.2% Brij 35, and 2 mM EDTA (pH 7.5), with a supplement of 2× protease inhibitor cocktail (Roche Diagnostics), 2× phosphatase inhibitor cocktail (Roche Diagnostics), 2 mM activated Na3VO4, and 2 mM PMSF. Lysates were centrifuged in a microfuge (14,000 rpm for 1 h at 4°C), and the supernatants were then collected and the remaining gel pellets were discarded as previously described (16). The supernatants were concentrated using a YM-10 Microcon centrifugal filter device (Millipore). Protein concentrations in supernatants were evaluated using a total protein assay (Bio-Rad). The protein samples were then boiled for 5 min after mixing with 4× sample buffer, containing 400 mM Tris·HCl, 8% SDS, 40% glycerol, 0.04% bromphenol blue, and 20% β-mercaptoethanol (pH 6.8), and stored at −80°C. Equal amounts of total protein per well were loaded onto 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked at room temperature with 2% enhanced chemiluminescence (ECL) advance blocking agent (Amersham, GE Healthcare) in TBS plus Tween-20 (16). The membranes were incubated overnight with a 1:1,000 dilution of a specific rabbit primary antibody (monoclonal antibodies: ERK1/2, phospho-ERK1/2, phospho-p38 MAPK, c-Fos, and c-Jun; and polyclonal antibodies: p38 MAPK, α-tubulin, and β-actin; all from Cell Signaling), followed by a 1.5-h room temperature incubation with an ECL horseradish peroxidase-linked anti-rabbit IgG antibody (Amersham, GE Healthcare). The proteins on polyvinylidene difluoride membranes were then detected using an ECL advanced Western blotting detection kit (Amersham, GE Healthcare) and the ChemiDoc XRS system with the Quantity One software (Bio-Rad). Some membranes were stripped using Restore Plus Western blot stripping buffer (Thermo Scientific Pierce) for a subsequent detection.
Nuclear protein extraction and AP-1 DNA binding activity assay.
Cells were released from collagen gels by 0.2% of collagenase I in growth medium and washed with ice-cold PBS as previously described (39). To extract nuclear protein, the cells were treated with NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific Pierce) by following the manufacturer's procedure. Because of the presence of EDTA, the extracts were mixed with CaCl2 to a final concentration of 5 mM to inactivate EDTA. Three micrograms of nuclear extract per well were used for an AP-1 DNA binding activity assay using the AP-1 transcription factor microplate assay (Marligen), following the manufacturer's procedure.
RNA extraction and gene expression analysis.
Total RNA was isolated from cells in collagen matrixes using TRIzol LS Reagent (Invitrogen) with the following modification. During RNA precipitation, high-salt (1.2 M Na-citrate/0.8 M NaCl) was added together with isopropanol. Under these conditions, RNA can be effectively precipitated while contaminating polysaccharides and proteoglycans remain in the soluble form (6). RNA samples were then converted to cDNA by reverse transcription (RT). For analyzing gene expression, the polymerase chain reaction (PCR) was performed using the following protocol: predenaturation at 95 °C for 5 min and then either 40 cycles (for MMP-1) or 30 cycles (for c-jun, c-fos, and GAPDH) of denaturation at 94 °C for 35 s, annealing at 52 °C for 35 s, and extension at 72 °C for 35 s, followed by a final extension at 72 °C for 10 min. The amplified products were separated by electrophoresis in 2.5% agarose gels and photographed under ultraviolet (UV) light in the presence of ethidium bromide. Quantitative real-time PCR (RT-qPCR) was also performed for c-fos, c-jun, and MMP-1 expression on the ABI PRISM 7000 sequence detection system (Applied Biosystems). GAPDH served as an internal control. The reactions were performed in 30-μl reaction mixture volumes containing SYBR Green PCR Master Mix (Applied Biosystems), cDNA, and specific primer pairs. RT-qPCR programs were set to 2 min at 50°C and 10 min at 95°C, followed by 45 cycles of 35 s at 95°C, 35 s at 52°C, and 40 s at 72°C. Following each PCR, a dissociation curve analysis was used to assess the specificity of product amplification. Primer sequences are listed in Table 1.
To silence ERK1/2, two ERK1 short hairpin (sh)RNAs and two ERK2 shRNAs, which were subcloned into pSUPER vector (kindly donated by Dr. Michal Hetman), were cotransfected into SMCs. The ERK1/2 shRNA target sequences were as follows: shERK1-1, GACCGGATGTTAACCTTTA; shERK1-2, ATGTCATAGGCATCCGAGA; shERK2-1, GTACAGAGCTCCAGAAATT; and shERK2–2, AGTTCGAGTTGCTATCAAG (21). To silence c-jun and c-fos, two c-jun shRNAs and c-fos shRNAs, which were subcloned into BS/U6 vector (kindly donated by Dr. Mingtao Li), were cotransfected into cells. The c-jun shRNA target sequences were as follows: shc-jun-a, ACAGGTGGCACAGCTTAAA; and shc-jun-b, AGTCATGAACCACGTTAAC. The c-fos shRNA target sequences were as follows: shc-fos-a, GGAGACAGATCAACTTGAA; and shc-fos-b, GCTGAAGGCTGAACCCTTT (27, 47). The transfections were conducted using Lipofectamine LTX and PLUS reagents (Invitrogen).
Results are presented as means ± SE. Data sets were analyzed for statistical significance using a Student's t-test with a two-tailed distribution, and P < 0.05 was considered statistically significant. For comparison of more than two groups, we used one-way ANOVA followed by the t-test with Bonferroni correction, and P < 0.05/N (N stands for number of comparisons) was considered statistically significant.
Interstitial flow induces SMC migration and MMP-1 expression, whereas PD-98059 abolishes the flow effects.
MAPKs are important mediators that regulate a variety of cellular processes, including gene expression, proliferation, survival, apoptosis, migration, and differentiation (36). Therefore, the roles of ERK1/2 and p38 MAPK were examined in this study. As shown in Fig. 1, interstitial flow significantly induced vascular SMC migration by twofold and ERK1/2 inhibitor (PD-98059) completely abolished flow-induced migration. Surprisingly, p38 MAPK inhibitor SB-203580 markedly enhanced flow-induced migration. In no-flow control cases, PD-98059 did not affect SMC migration, whereas SB-203580 promoted SMC migration.
We have previously shown that interstitial flow-induced cell migration in collagen I gels is controlled by MMP-1 (39). Therefore, we examined the effects of interstitial flow and MAPK inhibitors on MMP-1 gene expression. As shown in Fig. 2, interstitial flow significantly induced MMP-1 gene expression in vascular SMCs, and PD-98059 completely abolished flow-induced MMP-1 expression, whereas SB-203580 markedly enhanced flow-induced MMP-1 expression. In no-flow control cases, PD-98059 inhibited MMP-1 expression and SB-203580 slightly promoted MMP-1 gene expression. The combination of SMC migration data and MMP-1 expression data suggests that interstitial flow-induced cell migration and MMP-1 expression are ERK1/2 dependent, whereas p38 MAPK appears to play a downregulatory role.
Interstitial flow induces ERK1/2 and p38 MAPK activation.
To further investigate whether the interstitial flow and MAPK inhibitors really affect ERK1/2 activation, Western blot analysis was used to analyze the levels of both phosphorylated and total ERK1/2 (shown in Fig. 3A). During 60 min of exposure to interstitial flow, ERK1/2 was markedly activated as the phosphorylated ERK1/2 level increased greatly. PD-98059 dramatically inhibited ERK1/2 activation for no-flow (0 min) and flow (15, 30, and 60 min) cases compared with the cases without an addition of PD-98059. Interestingly, p38 MAPK inhibitor SB-203580 significantly enhanced ERK1/2 activation for no-flow (0 min) and flow (15, 30, and 60 min) cases. This unexpected stimulatory role of SB-203580 on ERK1/2 activation has been reported elsewhere (14, 24, 35). Sixty minutes of interstitial flow, with or without SB-203580 or PD-98059, had no effect on total ERK1/2.
To investigate whether interstitial flow and MAPK inhibitors affect p38 MAPK activation, both phosphorylated and total p38 MAPKs were detected by Western blot analysis. As shown in Fig. 3B, interstitial flow markedly activated p38 MAPK. SB-203580 clearly inhibited flow-induced p38 MAPK activation, whereas PD-98059 did not affect p38 MAPK activation. Sixty minutes of interstitial flow, with or without SB-203580 or PD-98059, had no effect on total p38 MAPK.
Interstitial flow induces SMC migration and MMP-1 expression via ERK1/2.
Since the pharmacological inhibitors may also influence other protein kinases (10), to further confirm the predominant regulatory role of ERK1/2 in MMP-1 expression and cell migration, ERK1/2 shRNAs were transfected into cells. After the knocking down of ERK1/2, both interstitial flow-induced MMP-1 expression (Fig. 4A) and cell migration (Fig. 4B) were completely abolished. In addition, the migration in no-flow control case (vector) was barely dependent on ERK1/2 (Figs. 1 and 4B), which is consistent with our previous observation that the migration of SMCs in the no-flow control case is independent of MMP-1 (39).
Taken together, these data (cell migration, MMP-1 expression, MAPK activation and inhibition, and ERK1/2 gene knockdown) suggest that interstitial flow-induced cell migration and MMP-1 expression proceed through an ERK1/2-dependent pathway. ERK1/2 activation plays a much more direct and critical role in regulating MMP-1 expression than p38 MAPK does, although the exact role of p38 MAPK is not clear.
Interstitial flow induces c-jun and c-fos expression through ERK1/2 activation.
Transcription factors c-jun and c-fos are members of the AP-1 family, and these immediate-early genes are targets of the ERK1/2 MAPK pathway (11). To investigate whether interstitial flow-induced ERK1/2 phosphorylation affects AP-1 transcription factor (c-Jun and c-Fos) expression, we determined c-jun and c-fos gene expression by RT-qPCR and c-Jun and c-Fos protein expression by Western blot analysis. Interstitial flow dramatically induced c-jun and c-fos transient gene expression (Fig. 5A) within 15 min, and the elevated expression then returned to the control level by 60 min. PD-98059 attenuated both c-jun and c-fos transient gene expression to their control levels. In the presence of SB-203580, interstitial flow significantly induced the c-jun and c-fos gene expression that remained at a relatively high level at 60 min although the peak level was not as high as the induction by interstitial flow alone (Fig. 5A). The change of c-fos expression was more pronounced than that of c-jun.
Similar trends were observed for AP-1 protein expression (Fig. 5B). Interstitial flow markedly induced c-Jun and c-Fos protein expression within 60 min. PD-98059 significantly reduced flow-induced c-Jun and c-Fos expression. SB-203580 raised c-Jun and c-Fos expression for both no-flow (0 min) and flow cases, but there appeared to be little flow induction.
Interstitial flow induces AP-1 DNA binding activity depending on ERK1/2 activation.
To regulate target gene transcription, AP-1 transcription factors have to translocate from the cytoplasm into the nucleus and bind to specific sites on DNA (AP-1 site). As shown in Fig. 6, interstitial flow significantly increased AP-1 DNA binding activity by twofold, and PD-98059 completely abolished the flow-increased DNA binding activity, suggesting that the increased DNA binding activity is ERK1/2 dependent. After treatment with SB-203580, the flow-induced AP-1 DNA binding activity was only slightly, but not significantly, higher than the flow-only case (Fig. 6). This is not consistent with the fact that the MMP-1 gene expression was much higher in the case of flow with SB-203580 than in the flow-only case (Fig. 2). However, it is possible that the sustained expression of AP-1 transcription factor stimulated by SB-203580 (Fig. 5) provided a sustained high level of DNA binding activity which maintained the MMP-1 expression at a high level.
Interstitial flow-induced MMP-1 expression is mediated by c-jun, but not c-fos.
To further investigate whether ERK1/2 regulated MMP-1 expression directly through AP-1, c-jun and c-fos genes were silenced by their shRNAs. As shown in Fig. 7, after knocking down c-jun, the flow-induced MMP-1 expression was completely abolished. Although interstitial flow significantly induced c-fos expression (Fig. 5), surprisingly, the silencing of c-fos did not affect MMP-1 expression (Fig. 8). These data suggest that it was c-jun but not c-fos that mediated flow-induced MMP-1 expression through an ERK1/2-dependent pathway.
In the present study, we observed that interstitial flow activated both ERK1/2 and p38 MAPK, PD-98059 abolished flow-induced ERK1/2 activation, and in contrast, SB-203580 enhanced flow-induced ERK1/2 activation and abolished flow-induced p38 MAPK activation (Fig. 3). Interstitial flow also induced MMP-1 expression and SMC migration, and PD-98059 inhibited flow-induced MMP-1 expression and cell migration, whereas SB-203580 enhanced MMP-1 expression and cell migration although the activation of p38 MAPK was inhibited (Figs. 1 and 2). Silencing ERK1/2 completely abolished flow-induced MMP-1 expression and cell migration (Fig. 4). These findings indicate that interstitial flow-induced MMP-1 expression and cell migration proceed through an ERK1/2-dependent pathway. ERK1/2 activation plays a much more critical role in regulating flow-induced MMP-1 expression than p38 MAPK although the exact role of p38 MAPK remains unclear.
There is abundant evidence in the literature that the induction of MMP-1 expression is related to the activation of MAPKs such as ERK1/2 and/or p38 MAPK (5, 28, 33). The activation of ERK1/2 by chemical and physical factors such as mitogenic growth factors, proinflammatory cytokines, heat shock, UV light, and other factors often induces MMP-1 expression in many cell types (5, 7, 12, 15, 28, 31). In contrast, the activation of p38 MAPK has opposing effects on MMP-1 gene expression depending on the mode of induction (13). For example, MMP-1 expression in human skin fibroblasts induced by tumor necrosis factor-α is through AP-1-dependent transcriptional activation via the ERK1/2 pathway and AP-1-independent enhancement via p38α MAPK by mRNA stabilization (33); lipopolysaccharide-induced monocyte MMP-1 expression is regulated by both ERK1/2 and p38 MAPK (23), whereas PDGF-BB and arsenite suppress MMP-1 expression via p38 MAPK activation (13, 46). One study has demonstrated that p38α, one isoform of p38 MAPK, inhibits the ERK1/2 pathway (46). Thus, in our study, the cross talk between ERK1/2 and p38 MAPK might play a role in interstitial flow-induced MMP-1 expression. However, although the role of p38 MAPK remains unclear, our data strongly suggest that ERK1/2 plays a predominant regulatory role in flow-induced MMP-1 expression.
In cardiovascular health and disease, it has been shown that the ERK1/2 MAPK plays an important role (30). Increased ERK1/2 activation contributed to augmented vascular SMC proliferation and neointima formation with aging in a rabbit study (17). The rapid activation of ERK1/2 after balloon injury of the rat carotid artery may be associated with vascular SMC migration and proliferation in vivo (19, 22). Completely blocking ERK1/2 activation in balloon-injured carotid arteries can inhibit carotid neointima formation by suppressing SMC migration and proliferation after 2 wk (18). The evidence from in vitro studies has also shown that ERK1/2 activation plays a crucial role in cardiovascular cell proliferation, cell migration, and MMP-1 expression (7–9, 15, 28, 29, 32).
The activation of ERK1/2 MAPK can induce the expression and activation of many transcription factors, such as AP-1. AP-1 transcription factors have a variety of physiological and pathophysiological consequences through their regulation of target gene expression. The AP-1 DNA binding element plays a pivotal role in the regulation of MMP expression since AP-1 sites are present throughout the MMP promoters (4, 43). Jun proteins can form stable dimers that bind AP-1 DNA recognition elements (5′-TGAG/CTCA-3′). Fos proteins can bind DNA by forming heterodimers with Jun proteins (Jun:Fos) that are more stable than Jun:Jun dimers (37). The ERK MAPK cascade is likely involved in the induction of the c-fos gene and in forming the Jun:Fos heterodimer (37). In the present study, we observed that interstitial flow significantly promoted AP-1 (c-Jun and c-Fos) expression (Fig. 5). The increased expression and activation of c-Jun and c-Fos led to an elevated AP-1 DNA binding activity (Fig. 6), which could promote MMP-1 expression. Using an ERK1/2 specific inhibitor PD-98059, we further demonstrated that interstitial flow-induced AP-1 DNA binding activity is ERK1/2 dependent (Fig. 6). However, interestingly, RNA interference results suggest that it was c-jun but not c-fos that regulated MMP-1 expression (Figs. 7 and 8), although c-fos was definitely induced by flow.
Taken together, we described for the first time a mechanism of interstitial flow-induced MMP-1 expression in vascular SMCs in 3-D collagen I gels (shown in Fig. 9). Interstitial flow, by an as-yet to-be-determined mechanotransduction mechanism, can induce ERK1/2 MAPK activation, which promotes AP-1 transcription factor expression and AP-1 DNA binding activity, resulting in an elevated MMP-1 expression. Proteolysis of collagen by upregulated MMP-1 then facilitates SMC migration through collagen I. However, the mechanism by which vascular cells “sense” interstitial flow and then trigger ERK1/2 signaling pathway requires further investigation. With the consideration that cells in 3-D exhibit matrix adhesions all over their surface, cell-matrix adhesion and tethering through integrin-rich focal adhesions would be a potential mechanosensor (38, 39). Another candidate would be the cell surface glycocalyx, which has been shown to mediate fluid shear stress-regulated vascular SMC contraction (1) and various endothelial cell functions (42).
Transmural interstitial flow is elevated when the endothelium is injured because of chemical or mechanical stimuli or when the vascular hydraulic conductivity is enhanced by inflammation and hypertension. Thus elevated interstitial flow may be associated with vessel remodeling and neointima formation (34, 39). The present study reveals a mechanism whereby altered interstitial flow during the early stages of vascular injury stimulates MMP expression and vascular SMC and fibroblast migration, which can lead to neointima formation. These findings suggest that methods to control interstitial flow or directly inhibit MMP expression or suppress ERK1/2 MAPK activation may be able to limit neointima formation clinically after vascular injury.
This study was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-35549 and RO1-HL-57093.
No conflicts of interest are declared by the author(s).
We thank Dr. Yongzhong Hou for valuable discussions and Hui Wang for cell migration assay.
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