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Collagen stimulates discoidin domain receptor 1-mediated migration of smooth muscle cells through Src
Cardiovascular Pathology 20 (2011) 71 – 76
Original Article
Collagen stimulates discoidin domain receptor 1-mediated migration of smooth muscle cells through Src Katherine Kun Lu, Dan Trcka, Michelle P. Bendeck⁎ Department of Laboratory Medicine and Pathobiology and Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada Received 15 September 2009; received in revised form 6 November 2009; accepted 24 December 2009
Abstract Background: Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase which mediates the migration and proliferation of several cell types. DDR1 is expressed in vascular smooth muscle cells (SMCs) during atherosclerosis and following vascular injury, mediating cell migration and contributing to disease pathogenesis. However, very little is known about the signaling pathways activated by the DDR1 in SMCs. Therefore we have studied the involvement of Src and mitogen-activated protein kinase (MAPK) signaling pathways downstream of DDR1 in vascular SMCs. Methods: Cells harvested from DDR1−/−, DDR1+/+ mice, and DDR1+/+ cells overexpressing human DDR1b (O/hDDR1b) were used for these studies. Results: Stimulation of O/hDDR1b cells with type I collagen resulted in increased tyrosine phosphorylation of DDR1. The non-receptor kinase Src co-immunoprecipitated with DDR1, and the Src inhibitor PP2 inhibited type I collagen-induced tyrosine phosphorylation of DDR1. Stimulation of DDR1-expressing cells with collagen resulted in the activation of extracellular signal-regulated kinase 1/2 (ERK1/2); however, ERK1/2 was not activated in DDR1-deficient cells. By contrast, p38 MAPK (p38) was activated by collagen stimulation in both DDR1-expressing and DDR1-deficient cells. Treatment with PP2 attenuated DDR1-dependent ERK1/2 activation, but not p38 activation. Finally, treatment of SMCs with PP2, or the MEK inhibitor PD98059, inhibited migration toward type I collagen in a chemotaxis chamber. However, PP2 but not PD98059 had a greater effect in reducing the migration of DDR1+/+ cells compared to DDR1−/− cells, suggesting that Src but not ERK1/2 was important in regulating DDR1dependent SMC migration. Conclusions: Type I collagen induces SMC migration through DDR1 and this is mediated via Src signaling. © 2011 Elsevier Inc. All rights reserved. Keywords: Smooth muscle cell; Collagen; Migration; Atherosclerosis; Discoidin domain receptor 1
1. Introduction Smooth muscle cell (SMC) migration from the media to the intimal layer of the artery has been implicated in a variety of related cardiovascular diseases including atherosclerosis, Financial support: Funded by grants from the CIHR MOP37847 and the HSFO NA6096 and T6734 to M.B. M.B. was a career investigator of the HSFO. K.K.L was supported by a fellowship from the Heart and Stroke Richard Lewar Center at the University of Toronto. ⁎ Corresponding author. Medical Sciences Building, Room 6213, 1 King's College Circle, Toronto, ON, Canada M5S 1A8. Tel.: +1 416 946 7133; fax: +1 416 978 5959. E-mail address: [email protected] (M.P. Bendeck). 1054-8807/09/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2009.12.006
in-stent restenosis, and transplant vasculopathy. Cell migration requires properly coordinated interactions between cells and the extracellular matrix, and collagen is a known stimulus for SMC migration [1]. Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase which is activated by a wide range of collagens [2]. DDR1 is expressed by SMCs in atherosclerotic lesions at both early and advanced stages [3], and atherosclerotic plaque development is markedly attenuated after deletion of the Ddr1 gene in Ldlr-deficient mice [4]. Moreover, DDR1 expression is increased after balloon injury of the rat carotid artery, and following wire injury of the carotid artery, neointimal thickening is markedly reduced in Ddr1−/−
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compared to Ddr1 +/+ mice, secondary to decreased migration of Ddr1-deficient SMCs [5]. Activation of DDR1 leads to phosphorylation of tyrosine residues at several distinct sites in the cytoplasmic domain and to recruitment of the adapter molecules ShcA, Nck, and Shp-2 [6,7]. The non-receptor tyrosine kinase Src has also been implicated in mediating DDR1 phosphorylation [8] and receptor cleavage [9], although these studies did not demonstrate the association between DDR1 and Src. DDR1 exhibits unusually long activation kinetics; maximal activation is achieved between 1 and 18 h after collagen stimulation [2], with the activation time varying depending on the cell type, cell adhesion, and the activity of intracellular phosphatases [10]. In several cell types, DDR1 activates signaling through mitogen-activated protein kinase (MAPK) pathways; activation of a given family member may vary depending on the cell type, composition of the extracellular matrix, and adhesion status of the cell [11–14]. Despite some advances in understanding the signaling pathways triggered by DDR1 in various cell types, and an important role for DDR1 in controlling SMC migration after arterial injury, there is little known about DDR1 signaling in vascular SMCs. In previous studies, we have shown that DDR1 kinase activity and receptor phosphorylation are required for MMP production and SMC migration in response to collagen stimulation [15]; however, we did not define the downstream signaling pathways involved. Activation of MAPKs in SMCs mediates migration in response to several growth factors and cytokines [16–21]. However, the relationships between DDR1 and MAPK signaling in migrating SMCs have not been investigated. Herein we study intracellular signaling downstream of the DDR1 in SMCs and determine whether activation of these pathways is important in mediating SMC migration towards collagen.
Care. Mouse carotid SMCs were harvested from DDR1−/− and DDR1+/+ mice as previously described [5]. Cell cultures were maintained in DMEM+10% fetal bovine serum (FBS) at 37°C with 5% CO2. Cells between Passage 4 and Passage 10 were used for the experiments. In order to generate DDR1 overexpressing cells (O/ hDDR1b), DDR1+/+ SMCs were infected with a retrovirus encoding the human DDR1b isoform as previously described [15]. 2.3. Western blotting and immunoprecipitation Cell growth was arrested by incubation in DMEM+0.4% FBS for 16 h. The media was replaced by serum-free DMEM and cells were treated with type I collagen at 100 μg/ml for different time periods. In some experiments, cells were pretreated with 10 μM PP2 or PP3 for 30 min before type I collagen treatment. For immunoprecipitation, cells were harvested in RIPA buffer and cell lysates were centrifuged at 14,000×g for 5 min at 4°C to clear cell debris. Three hundred micrograms of total protein was used for immunoprecipitation. Cell lysates were incubated with primary antibody (2–4 μg per sample) for 1.5 h with rocking at 4°C. Then 20 μl of protein A-agarose beads was added to the sample and incubated for 16 h at 4°C with rocking. Immunocomplexes were captured by centrifugation at 2500 rpm for 5 min. Pellets were washed three times with 200 μl RIPA buffer. SDS sample buffer was added to the pellets after final wash, and samples were boiled at
2. Methods 2.1. Materials ERK1/2, p38, and Src antibodies were purchased from Cell Signaling Technology, Inc.; 4G10 (anti-phosphotyrosine antibody) from Upstate Biotechnology; DDR1 Cterminal antibody and protein-A agarose beads from Santa Cruz Biotechnology, Inc.; and β-actin antibody from Abcam. PureCol (type I collagen) was purchased from Inamed. Src inhibitor PP2 and negative control PP3 were purchased from Calbiochem. All other reagents were purchased from Sigma unless specified otherwise. 2.2. Cell culture Animal experiments were performed in accordance with the guidelines of the Canada Council on Animal
Fig. 1. Stimulation with type I collagen activates DDR1 phosphorylation. O/ hDDR1b SMCs were treated with 100 μg/ml soluble type I collagen, followed by immunoprecipitation of DDR1 and Western blotting for phosphotyrosine. Full-length DDR1b (125 kDa) and the C-terminal cleavage product of DDR1b (62 kDa; β subunit) are evident on the blots. The level of phosphorylated DDR1 was normalized to total DDR1 and expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
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95°C for 5 min. Then the immunoprecipitated material was used for Western blot analysis. Western blots were analyzed by scanning densitometry. Each experiment was repeated at least four times.
3. Results
2.4. Migration assay
Human DDR1b was overexpressed in mouse carotid SMCs (O/hDDR1b) to facilitate immunoprecipitation and detection of the receptor. DDR1 activation is characterized by tyrosine phosphorylation within the cytoplasmic domain of the receptor. Following type I collagen stimulation of O/ hDDR1b cells, DDR1 tyrosine phosphorylation was evident at 90 min and peaked at 16 h (Fig. 1). Bands were present representing the phosphorylated full-length receptor (125 kDa), as well as the C-terminal fragment of the receptor (β subunit, 62 kDa), which is generated by proteolytic cleavage after activation [9,22]
SMC migration was assayed using a chemotaxis chamber assay. The cell inserts containing polycarbonate membranes with 8-μm pores (Becton Dickinson) were placed in a 24-well tissue culture plate to generate the upper and lower chambers. Sixty-five thousand DDR1−/− or DDR1+/+ cells suspended in 0.25 ml DMEM containing 200 μg/ml BSA (DMEM/BSA) were placed in the top chamber. The lower chamber was filled with 0.5 ml DMEM/BSA with 100 μg/ml type I collagen. In experiments where inhibitors were used, Src (PP2) or MEK (PD98059) inhibitor (to inhibit ERK1/2 activation) was added to a final concentration of 10 μM to both the top and the bottom of the chamber for the duration of the experiment. Cells were allowed to migrate for 6 h. Migrated cells on the bottom of the filter were counted in four microscopic fields taken at 100× magnification, and the data was averaged. n=7–12 for each group. Statistical analysis was performed using two-way ANOVA followed by Holm–Sidak test to make pairwise comparisons (Sigma Stat).
3.1. Tyrosine phosphorylation of DDR1 in response to type I collagen stimulation
3.2. Src is involved in collagen-dependent phosphorylation of DDR1 Previously, it was shown that Src regulates DDR1 tyrosine phosphorylation in mammary epithelial cells [8]. Therefore we reasoned that Src might be similarly involved in DDR1 activation in SMCs. Using an antibody to immunoprecipitate Src, we determined that DDR1 coimmunoprecipitated with Src after stimulation of the cells with type I collagen (Fig. 2A). To determine whether Src was
Fig. 2. Src associates with and is involved in the phosphorylation of DDR1. (A) O/hDDR1b SMCs were untreated or treated with 100 μg/ml soluble type I collagen for 4 h, after which Src was immunoprecipitated then blots probed for DDR1 and Src. (B) O/hDDR1b SMCs were pretreated with the Src inhibitor PP2 (10 μM) or PP3 (10 μM) (a negative control for PP2) for 30 min, then 100 μg/ml type I collagen was added for another 4 h, followed by DDR1 immunoprecipitation, then blots were probed for phosphotyrosine and DDR1. The level of phosphorylated DDR1 was normalized to total DDR1 and expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
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but activation of p38 occurs independent of DDR1 in collagen stimulated SMCs. 3.4. Src is involved in DDR1-dependent ERK1/2 phosphorylation We next determined whether Src was involved in collagen-stimulated MAPK activation in SMCs. ERK1/2 phosphorylation was detected in lysates from O/hDDR1b SMCs after 4 or 16 h of type I collagen treatment (Fig. 4). Src inhibition with PP2 reduced ERK1/2 phosphorylation, while the negative control PP3 had no effect (Fig. 4). Type I collagen treatment also led to the activation of p38; however, PP2 did not affect the phosphorylation of p38 (Fig. 4). These data show that Src is important for ERK1/2 activation, but not for p38 activation, in response to type I collagen stimulation of SMCs.
Fig. 3. Stimulation with type I collagen activates DDR1-dependent ERK1/2 activation and DDR1-independent p38 activation. DDR1−/−, DDR1+/+, and O/hDDR1b SMCs were treated with 100 μg/ml type I collagen for 4 or 16 h. Cell lysates were run on Western blots and probed with the antibodies indicated. The levels of phosphorylated ERK1/2 and p38 were normalized to their respective total proteins and were expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
required for DDR1 activation, we treated SMCs with the Src inhibitor PP2 and found that DDR1 phosphorylation was dramatically reduced by PP2 treatment (Fig. 2B). By contrast, PP3, a negative control which does not inhibit Src, did not affect DDR1 phosphorylation (Fig. 2B). 3.3. Type I collagen induced DDR1-dependent phosphorylation of ERK1/2 and DDR1-independent phosphorylation of p38 We next asked whether type I collagen stimulation of SMCs resulted in the activation of MAPKs. Type I collagen was used to stimulate DDR1−/−, DDR1+/+, and O/hDDR1b SMCs, and the cell lysates were assayed for ERK1/2 and p38 activation by Western blotting (Fig. 3). Treatment with type I collagen led to an increase in the phosphorylation of ERK1/2 which was evident at 16 h in DDR1+/+ and O/hDDR1b SMCs (Fig. 3). By contrast, ERK1/2 was not activated in DDR1−/− SMCs. P38 was activated by collagen treatment of the cells whether DDR1 was present or absent. The data show that activation of ERK1/2 is dependent upon DDR1,
Fig. 4. Src is involved in DDR1-dependent ERK1/2 phosphorylation. O/ hDDR1b SMCs were pretreated with either the Src inhibitor PP2 (10 μM) or PP3 (10 μM) (a negative control for PP2) for 30 min, then stimulated with 100 μg/ml type I collagen for 4 or 16 h. Cell lysates were run on Western blots and probed with the antibodies indicated. The levels of phosphorylated ERK1/2 and p38 were normalized to their respective total proteins and were expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
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Fig. 5. Src is involved in collagen-stimulated DDR1-dependent SMC migration. DDR1−/− and DDR1+/+ SMCs were subjected to chemotaxis chamber assays stimulated with 100 μg/ml type I collagen added to the lower chamber. In some experiments the collagen-stimulated cells were also treated with 10 μM Src inhibitor PP2 or 10 μM MEK inhibitor PD98095. ⁎Pb.05 comparing DDR1+/+ to DDR1−/− cells, both with collagen stimulation. † Pb.05 compared to the same cell type stimulated with collagen only.
3.5. Src is involved in collagen-stimulated DDR1-dependent SMC migration SMC migration was assessed using chemotaxis chambers with type I collagen suspended in the bottom of the chamber as a chemoattractant. Collagen stimulated the migration of both DDR1+/+ and DDR1−/− SMCs, and migration was significantly greater in DDR1+/+ compared to DDR1−/− SMCs (Fig. 5). Treatment with the Src inhibitor PP2 reduced migration in both DDR1−/− and DDR1+/+ SMCs, with a trend toward greater inhibition observed in the DDR1+/+ cells (Fig. 5). Treatment with PD98059 to inhibit the activation of ERK1/2 significantly reduced the migration of both cell types, but there was no difference in migration between DDR1−/− and DDR1+/+ cells (Fig. 5). This data indicates that DDR1 signals through Src, but not necessarily through ERK1/2 to control SMC migration in response to collagen. 4. Discussion In previous work using mouse models of atherosclerosis and restenosis, we were the first to show that DDR1 is important for cell migration; DDR1−/− SMCs migrated less compared to DDR1+/+ SMCs after stimulation with either type I or type VIII collagen [5,15]. As a consequence of decreased migration and proliferation in DDR1-deficient SMCs, there was a dramatic decrease in intimal thickening after carotid injury in DDR1−/− mice compared to Ddr1+/+
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Fig. 6. Proposed model of DDR1 signaling in SMCs. In response to stimulation with type I collagen, DDR1 is tyrosine phosphorylated and the non-receptor tyrosine kinase Src associates with DDR1. Src regulates DDR1 phosphorylation and the downstream activation of ERK1/2. Src activates DDR1-dependent SMC migration, although this does not appear to depend on ERK1/2. Type I collagen also stimulates p38 activation, but this is not dependent upon DDR1.
mice [5]. In the current study, we begin to elucidate the signaling mechanism for DDR1-mediated SMC migration. In response to type I collagen stimulation, DDR1 is tyrosine phosphorylated within the kinase domain. Src regulates DDR1 phosphorylation and downstream signaling via ERK1/2 and influences cell migration. A proposed model for our studies can be found in Fig. 6. Previous studies in other cell types have suggested that the non-receptor tyrosine kinase Src modulates DDR1 function. Src regulates DDR1 tyrosine phosphorylation in mammary epithelial cells [8] and is involved in shedding of the DDR1 ectodomain in breast carcinoma cells [9]. Our current studies show that Src is associated with DDR1 in vascular SMCs; however, the co-immunoprecipitation experiments suggest that only a small fraction of the pool of DDR1 binds to Src. Nevertheless, inhibition of Src activity reduces the phosphorylation of DDR1. Consequently, this Src/DDR1 association has a great influence on DDR1 tyrosine phosphorylation. We have also shown that DDR1 is required for the collagen-stimulated activation of ERK1/2 in SMCs. By contrast, collagen stimulates p38 signaling even in the absence of DDR1. Furthermore, the Src inhibitor PP2 inhibited the activation of ERK1/2 in SMCs, but did not inhibit p38 activation. This suggests that Src activates ERK1/ 2 pathways downstream of DDR1, whereas activation of p38 likely occurs via integrin signaling and is independent of Src. This is consistent with results from studies that have shown that integrin receptor signaling activates p38, which is important in mediating cell migration and contraction of three-dimensional collagen matrices [23].
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Our results add to a body of literature which reveals that there is substantial variation in DDR1 signaling. For example, in mesangial cells, DDR1 suppresses ERK1/2 activation to maintain cell quiescence [11], while in mammary epithelial cells DDR1 stimulation leads to ERK1/2 activation and cell proliferation [12]. In macrophages, DDR1b promotes activation of p38 [24], ERK1/2, and JNK [13]. DDR1 also signals via JNK to mediate the epithelial to mesenchymal transition of pancreatic cancer cells [14]. The context in which a cell is exposed to the extracellular matrix may also be very important; when DDR1 is overexpressed in Madin–Darby canine kidney cells grown in three-dimensional collagen, migration is inhibited [25]. Future studies are needed to determine whether there are differences in the expression of receptor isoforms or differences in the type or the physical state of collagen that signal via DDR1. In SMCs, migration in response to collagen was critically dependent upon DDR1, as DDR1+/+ cells migrated more than DDR1−/− cells. Migration was also dependent upon Src activity, as PP2 treatment decreased the migration of both DDR1+/+ and DDR1−/− cells. Furthermore, Src inhibition had a greater effect in attenuating the migration of DDR1+/+ SMCs, suggesting that Src is downstream of DDR1 in regulating migration. The MEK inhibitor also inhibited cell migration, although not as strongly as the Src inhibitor, and there was no difference in migration dependent upon DDR1 expression. This suggests that although activation of ERK1/2 in response to collagen stimulation is dependent upon DDR1, ERK1/2 does not mediate DDR1-dependent differences in cell migration. Instead, other factors downstream of Src must be involved, and this will require further study. Taken together, our data indicates that collagen stimulates SMC migration which is mediated through DDR1 activating Src downstream of the receptor. References [1] Adiguzel E, Ahmad PJ, Franco C, Bendeck MP. Collagens in the progression and complications of atherosclerosis. Vasc Med 2009;14: 73–89. [2] Vogel W, Gish GD, Alves F, Pawson T. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol Cell 1997;1:13–23. [3] Ferri N, Carragher NO, Raines EW. Role of discoidin domain receptors 1 and 2 in human smooth muscle cell-mediated collagen remodeling: potential implications in atherosclerosis and lymphangioleiomyomatosis. Am J Pathol 2004;164:1575–85. [4] Franco C, Hou G, Ahmad PJ, Fu EY, Koh L, Vogel WF, et al. Discoidin domain receptor 1 (Ddr1) deletion decreases atherosclerosis by accelerating matrix accumulation and reducing inflammation in low-density lipoprotein receptor deficient mice. Circ Res 2008;102: 1202–11. [5] Hou G, Vogel W, Bendeck MP. The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair. J Clin Invest 2001;107: 727–35. [6] Koo DH, McFadden C, Huang Y, Abdulhussein R, Friese-Hamim M, Vogel WF. Pinpointing phosphotyrosine-dependent interactions downstream of the collagen receptor DDR1. FEBS Lett 2006;580: 15–22.
[7] Wang CZ, Su HW, Hsu YC, Shen MR, Tang MJ. A discoidin domain receptor 1/SHP-2 signaling complex inhibits alpha2beta1integrin-mediated signal transducers and activators of transcription 1/3 activation and cell migration. Mol Biol Cell 2006;17: 2839–52. [8] Dejmek J, Dib K, Jonsson M, Andersson T. Wnt-5a and G-protein signaling are required for collagen-induced DDR1 receptor activation and normal mammary cell adhesion. Int J Cancer 2003;103:344–51. [9] Slack BE, Siniaia MS, Blusztajn JK, Collagen type I. Selectively activates ectodomain shedding of the discoidin domain receptor 1: involvement of Src tyrosine kinase. J Cell Biochem 2006. [10] L'hote CG, Thomas PH, Ganesan TS. Functional analysis of discoidin domain receptor 1: effect of adhesion on DDR1 phosphorylation. FASEB J 2002;16:234–6. [11] Curat CA, Vogel WF. Discoidin domain receptor 1 controls growth and adhesion of mesangial cells. J Am Soc Nephrol 2002;13: 2648–56. [12] Hilton HN, Stanford PM, Harris J, Oakes SR, Kaplan W, Daly RJ, et al. KIBRA interacts with discoidin domain receptor 1 to modulate collagen-induced signalling. Biochim Biophys Acta 2008;1783: 383–93. [13] Kim SH, Lee S, Suk K, Bark H, Jun CD, Kim DK, et al. Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Free Radic Biol Med 2007;42: 343–52. [14] Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, Johnson KR. Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1. J Cell Biol 2008;180:1277–89. [15] Hou G, Vogel WF, Bendeck MP. Tyrosine kinase activity of discoidin domain receptor 1 is necessary for smooth muscle cell migration and matrix metalloproteinase expression. Circ Res 2002;90:1147–9. [16] Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 2001;410:37–40. [17] Pearson G, Robinson F, Beers GT, Xu BE, Karandikar M, Berman K, et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001;22:153–83. [18] Xi XP, Graf K, Goetze S, Fleck E, Hsueh WA, Law RE. Central role of the MAPK pathway in ang II-mediated DNA synthesis and migration in rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1999;19:73–82. [19] Yamboliev IA, Gerthoffer WT. Modulatory role of ERK MAPKcaldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs. Am J Physiol Cell Physiol 2001;280: C1680–8. [20] Wang Z, Castresana MR, Newman WH. Reactive oxygen speciessensitive p38 MAPK controls thrombin-induced migration of vascular smooth muscle cells. J Mol Cell Cardiol 2004;36:49–56. [21] Di LG, Pradhan S, Dhadwal AK, Chen A, Ueno H, Sumpio BE. Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration. Atherosclerosis 2005;178: 271–7. [22] Vogel WF. Ligand-induced shedding of discoidin domain receptor 1. FEBS Lett 2002;514:175–80. [23] Li S, Moon JJ, Miao H, Jin G, Chen BP, Yuan S, et al. Signal transduction in matrix contraction and the migration of vascular smooth muscle cells in three-dimensional matrix. J Vasc Res 2003;40: 378–88. [24] Matsuyama W, Wang L, Farrar WL, Faure M, Yoshimura T. Activation of discoidin domain receptor 1 isoform b with collagen up-regulates chemokine production in human macrophages: role of p38 mitogen-activated protein kinase and NF-kappa B. J Immunol 2004;172:2332–40. [25] Wang CZ, Hsu YM, Tang MJ. Function of discoidin domain receptor I in HGF-induced branching tubulogenesis of MDCK cells in collagen gel. J Cell Physiol 2004.
Original Article
Collagen stimulates discoidin domain receptor 1-mediated migration of smooth muscle cells through Src Katherine Kun Lu, Dan Trcka, Michelle P. Bendeck⁎ Department of Laboratory Medicine and Pathobiology and Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada Received 15 September 2009; received in revised form 6 November 2009; accepted 24 December 2009
Abstract Background: Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase which mediates the migration and proliferation of several cell types. DDR1 is expressed in vascular smooth muscle cells (SMCs) during atherosclerosis and following vascular injury, mediating cell migration and contributing to disease pathogenesis. However, very little is known about the signaling pathways activated by the DDR1 in SMCs. Therefore we have studied the involvement of Src and mitogen-activated protein kinase (MAPK) signaling pathways downstream of DDR1 in vascular SMCs. Methods: Cells harvested from DDR1−/−, DDR1+/+ mice, and DDR1+/+ cells overexpressing human DDR1b (O/hDDR1b) were used for these studies. Results: Stimulation of O/hDDR1b cells with type I collagen resulted in increased tyrosine phosphorylation of DDR1. The non-receptor kinase Src co-immunoprecipitated with DDR1, and the Src inhibitor PP2 inhibited type I collagen-induced tyrosine phosphorylation of DDR1. Stimulation of DDR1-expressing cells with collagen resulted in the activation of extracellular signal-regulated kinase 1/2 (ERK1/2); however, ERK1/2 was not activated in DDR1-deficient cells. By contrast, p38 MAPK (p38) was activated by collagen stimulation in both DDR1-expressing and DDR1-deficient cells. Treatment with PP2 attenuated DDR1-dependent ERK1/2 activation, but not p38 activation. Finally, treatment of SMCs with PP2, or the MEK inhibitor PD98059, inhibited migration toward type I collagen in a chemotaxis chamber. However, PP2 but not PD98059 had a greater effect in reducing the migration of DDR1+/+ cells compared to DDR1−/− cells, suggesting that Src but not ERK1/2 was important in regulating DDR1dependent SMC migration. Conclusions: Type I collagen induces SMC migration through DDR1 and this is mediated via Src signaling. © 2011 Elsevier Inc. All rights reserved. Keywords: Smooth muscle cell; Collagen; Migration; Atherosclerosis; Discoidin domain receptor 1
1. Introduction Smooth muscle cell (SMC) migration from the media to the intimal layer of the artery has been implicated in a variety of related cardiovascular diseases including atherosclerosis, Financial support: Funded by grants from the CIHR MOP37847 and the HSFO NA6096 and T6734 to M.B. M.B. was a career investigator of the HSFO. K.K.L was supported by a fellowship from the Heart and Stroke Richard Lewar Center at the University of Toronto. ⁎ Corresponding author. Medical Sciences Building, Room 6213, 1 King's College Circle, Toronto, ON, Canada M5S 1A8. Tel.: +1 416 946 7133; fax: +1 416 978 5959. E-mail address: [email protected] (M.P. Bendeck). 1054-8807/09/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2009.12.006
in-stent restenosis, and transplant vasculopathy. Cell migration requires properly coordinated interactions between cells and the extracellular matrix, and collagen is a known stimulus for SMC migration [1]. Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase which is activated by a wide range of collagens [2]. DDR1 is expressed by SMCs in atherosclerotic lesions at both early and advanced stages [3], and atherosclerotic plaque development is markedly attenuated after deletion of the Ddr1 gene in Ldlr-deficient mice [4]. Moreover, DDR1 expression is increased after balloon injury of the rat carotid artery, and following wire injury of the carotid artery, neointimal thickening is markedly reduced in Ddr1−/−
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compared to Ddr1 +/+ mice, secondary to decreased migration of Ddr1-deficient SMCs [5]. Activation of DDR1 leads to phosphorylation of tyrosine residues at several distinct sites in the cytoplasmic domain and to recruitment of the adapter molecules ShcA, Nck, and Shp-2 [6,7]. The non-receptor tyrosine kinase Src has also been implicated in mediating DDR1 phosphorylation [8] and receptor cleavage [9], although these studies did not demonstrate the association between DDR1 and Src. DDR1 exhibits unusually long activation kinetics; maximal activation is achieved between 1 and 18 h after collagen stimulation [2], with the activation time varying depending on the cell type, cell adhesion, and the activity of intracellular phosphatases [10]. In several cell types, DDR1 activates signaling through mitogen-activated protein kinase (MAPK) pathways; activation of a given family member may vary depending on the cell type, composition of the extracellular matrix, and adhesion status of the cell [11–14]. Despite some advances in understanding the signaling pathways triggered by DDR1 in various cell types, and an important role for DDR1 in controlling SMC migration after arterial injury, there is little known about DDR1 signaling in vascular SMCs. In previous studies, we have shown that DDR1 kinase activity and receptor phosphorylation are required for MMP production and SMC migration in response to collagen stimulation [15]; however, we did not define the downstream signaling pathways involved. Activation of MAPKs in SMCs mediates migration in response to several growth factors and cytokines [16–21]. However, the relationships between DDR1 and MAPK signaling in migrating SMCs have not been investigated. Herein we study intracellular signaling downstream of the DDR1 in SMCs and determine whether activation of these pathways is important in mediating SMC migration towards collagen.
Care. Mouse carotid SMCs were harvested from DDR1−/− and DDR1+/+ mice as previously described [5]. Cell cultures were maintained in DMEM+10% fetal bovine serum (FBS) at 37°C with 5% CO2. Cells between Passage 4 and Passage 10 were used for the experiments. In order to generate DDR1 overexpressing cells (O/ hDDR1b), DDR1+/+ SMCs were infected with a retrovirus encoding the human DDR1b isoform as previously described [15]. 2.3. Western blotting and immunoprecipitation Cell growth was arrested by incubation in DMEM+0.4% FBS for 16 h. The media was replaced by serum-free DMEM and cells were treated with type I collagen at 100 μg/ml for different time periods. In some experiments, cells were pretreated with 10 μM PP2 or PP3 for 30 min before type I collagen treatment. For immunoprecipitation, cells were harvested in RIPA buffer and cell lysates were centrifuged at 14,000×g for 5 min at 4°C to clear cell debris. Three hundred micrograms of total protein was used for immunoprecipitation. Cell lysates were incubated with primary antibody (2–4 μg per sample) for 1.5 h with rocking at 4°C. Then 20 μl of protein A-agarose beads was added to the sample and incubated for 16 h at 4°C with rocking. Immunocomplexes were captured by centrifugation at 2500 rpm for 5 min. Pellets were washed three times with 200 μl RIPA buffer. SDS sample buffer was added to the pellets after final wash, and samples were boiled at
2. Methods 2.1. Materials ERK1/2, p38, and Src antibodies were purchased from Cell Signaling Technology, Inc.; 4G10 (anti-phosphotyrosine antibody) from Upstate Biotechnology; DDR1 Cterminal antibody and protein-A agarose beads from Santa Cruz Biotechnology, Inc.; and β-actin antibody from Abcam. PureCol (type I collagen) was purchased from Inamed. Src inhibitor PP2 and negative control PP3 were purchased from Calbiochem. All other reagents were purchased from Sigma unless specified otherwise. 2.2. Cell culture Animal experiments were performed in accordance with the guidelines of the Canada Council on Animal
Fig. 1. Stimulation with type I collagen activates DDR1 phosphorylation. O/ hDDR1b SMCs were treated with 100 μg/ml soluble type I collagen, followed by immunoprecipitation of DDR1 and Western blotting for phosphotyrosine. Full-length DDR1b (125 kDa) and the C-terminal cleavage product of DDR1b (62 kDa; β subunit) are evident on the blots. The level of phosphorylated DDR1 was normalized to total DDR1 and expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
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95°C for 5 min. Then the immunoprecipitated material was used for Western blot analysis. Western blots were analyzed by scanning densitometry. Each experiment was repeated at least four times.
3. Results
2.4. Migration assay
Human DDR1b was overexpressed in mouse carotid SMCs (O/hDDR1b) to facilitate immunoprecipitation and detection of the receptor. DDR1 activation is characterized by tyrosine phosphorylation within the cytoplasmic domain of the receptor. Following type I collagen stimulation of O/ hDDR1b cells, DDR1 tyrosine phosphorylation was evident at 90 min and peaked at 16 h (Fig. 1). Bands were present representing the phosphorylated full-length receptor (125 kDa), as well as the C-terminal fragment of the receptor (β subunit, 62 kDa), which is generated by proteolytic cleavage after activation [9,22]
SMC migration was assayed using a chemotaxis chamber assay. The cell inserts containing polycarbonate membranes with 8-μm pores (Becton Dickinson) were placed in a 24-well tissue culture plate to generate the upper and lower chambers. Sixty-five thousand DDR1−/− or DDR1+/+ cells suspended in 0.25 ml DMEM containing 200 μg/ml BSA (DMEM/BSA) were placed in the top chamber. The lower chamber was filled with 0.5 ml DMEM/BSA with 100 μg/ml type I collagen. In experiments where inhibitors were used, Src (PP2) or MEK (PD98059) inhibitor (to inhibit ERK1/2 activation) was added to a final concentration of 10 μM to both the top and the bottom of the chamber for the duration of the experiment. Cells were allowed to migrate for 6 h. Migrated cells on the bottom of the filter were counted in four microscopic fields taken at 100× magnification, and the data was averaged. n=7–12 for each group. Statistical analysis was performed using two-way ANOVA followed by Holm–Sidak test to make pairwise comparisons (Sigma Stat).
3.1. Tyrosine phosphorylation of DDR1 in response to type I collagen stimulation
3.2. Src is involved in collagen-dependent phosphorylation of DDR1 Previously, it was shown that Src regulates DDR1 tyrosine phosphorylation in mammary epithelial cells [8]. Therefore we reasoned that Src might be similarly involved in DDR1 activation in SMCs. Using an antibody to immunoprecipitate Src, we determined that DDR1 coimmunoprecipitated with Src after stimulation of the cells with type I collagen (Fig. 2A). To determine whether Src was
Fig. 2. Src associates with and is involved in the phosphorylation of DDR1. (A) O/hDDR1b SMCs were untreated or treated with 100 μg/ml soluble type I collagen for 4 h, after which Src was immunoprecipitated then blots probed for DDR1 and Src. (B) O/hDDR1b SMCs were pretreated with the Src inhibitor PP2 (10 μM) or PP3 (10 μM) (a negative control for PP2) for 30 min, then 100 μg/ml type I collagen was added for another 4 h, followed by DDR1 immunoprecipitation, then blots were probed for phosphotyrosine and DDR1. The level of phosphorylated DDR1 was normalized to total DDR1 and expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
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but activation of p38 occurs independent of DDR1 in collagen stimulated SMCs. 3.4. Src is involved in DDR1-dependent ERK1/2 phosphorylation We next determined whether Src was involved in collagen-stimulated MAPK activation in SMCs. ERK1/2 phosphorylation was detected in lysates from O/hDDR1b SMCs after 4 or 16 h of type I collagen treatment (Fig. 4). Src inhibition with PP2 reduced ERK1/2 phosphorylation, while the negative control PP3 had no effect (Fig. 4). Type I collagen treatment also led to the activation of p38; however, PP2 did not affect the phosphorylation of p38 (Fig. 4). These data show that Src is important for ERK1/2 activation, but not for p38 activation, in response to type I collagen stimulation of SMCs.
Fig. 3. Stimulation with type I collagen activates DDR1-dependent ERK1/2 activation and DDR1-independent p38 activation. DDR1−/−, DDR1+/+, and O/hDDR1b SMCs were treated with 100 μg/ml type I collagen for 4 or 16 h. Cell lysates were run on Western blots and probed with the antibodies indicated. The levels of phosphorylated ERK1/2 and p38 were normalized to their respective total proteins and were expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
required for DDR1 activation, we treated SMCs with the Src inhibitor PP2 and found that DDR1 phosphorylation was dramatically reduced by PP2 treatment (Fig. 2B). By contrast, PP3, a negative control which does not inhibit Src, did not affect DDR1 phosphorylation (Fig. 2B). 3.3. Type I collagen induced DDR1-dependent phosphorylation of ERK1/2 and DDR1-independent phosphorylation of p38 We next asked whether type I collagen stimulation of SMCs resulted in the activation of MAPKs. Type I collagen was used to stimulate DDR1−/−, DDR1+/+, and O/hDDR1b SMCs, and the cell lysates were assayed for ERK1/2 and p38 activation by Western blotting (Fig. 3). Treatment with type I collagen led to an increase in the phosphorylation of ERK1/2 which was evident at 16 h in DDR1+/+ and O/hDDR1b SMCs (Fig. 3). By contrast, ERK1/2 was not activated in DDR1−/− SMCs. P38 was activated by collagen treatment of the cells whether DDR1 was present or absent. The data show that activation of ERK1/2 is dependent upon DDR1,
Fig. 4. Src is involved in DDR1-dependent ERK1/2 phosphorylation. O/ hDDR1b SMCs were pretreated with either the Src inhibitor PP2 (10 μM) or PP3 (10 μM) (a negative control for PP2) for 30 min, then stimulated with 100 μg/ml type I collagen for 4 or 16 h. Cell lysates were run on Western blots and probed with the antibodies indicated. The levels of phosphorylated ERK1/2 and p38 were normalized to their respective total proteins and were expressed relative to controls not stimulated with collagen. Data are presented as mean±S.E.M.
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Fig. 5. Src is involved in collagen-stimulated DDR1-dependent SMC migration. DDR1−/− and DDR1+/+ SMCs were subjected to chemotaxis chamber assays stimulated with 100 μg/ml type I collagen added to the lower chamber. In some experiments the collagen-stimulated cells were also treated with 10 μM Src inhibitor PP2 or 10 μM MEK inhibitor PD98095. ⁎Pb.05 comparing DDR1+/+ to DDR1−/− cells, both with collagen stimulation. † Pb.05 compared to the same cell type stimulated with collagen only.
3.5. Src is involved in collagen-stimulated DDR1-dependent SMC migration SMC migration was assessed using chemotaxis chambers with type I collagen suspended in the bottom of the chamber as a chemoattractant. Collagen stimulated the migration of both DDR1+/+ and DDR1−/− SMCs, and migration was significantly greater in DDR1+/+ compared to DDR1−/− SMCs (Fig. 5). Treatment with the Src inhibitor PP2 reduced migration in both DDR1−/− and DDR1+/+ SMCs, with a trend toward greater inhibition observed in the DDR1+/+ cells (Fig. 5). Treatment with PD98059 to inhibit the activation of ERK1/2 significantly reduced the migration of both cell types, but there was no difference in migration between DDR1−/− and DDR1+/+ cells (Fig. 5). This data indicates that DDR1 signals through Src, but not necessarily through ERK1/2 to control SMC migration in response to collagen. 4. Discussion In previous work using mouse models of atherosclerosis and restenosis, we were the first to show that DDR1 is important for cell migration; DDR1−/− SMCs migrated less compared to DDR1+/+ SMCs after stimulation with either type I or type VIII collagen [5,15]. As a consequence of decreased migration and proliferation in DDR1-deficient SMCs, there was a dramatic decrease in intimal thickening after carotid injury in DDR1−/− mice compared to Ddr1+/+
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Fig. 6. Proposed model of DDR1 signaling in SMCs. In response to stimulation with type I collagen, DDR1 is tyrosine phosphorylated and the non-receptor tyrosine kinase Src associates with DDR1. Src regulates DDR1 phosphorylation and the downstream activation of ERK1/2. Src activates DDR1-dependent SMC migration, although this does not appear to depend on ERK1/2. Type I collagen also stimulates p38 activation, but this is not dependent upon DDR1.
mice [5]. In the current study, we begin to elucidate the signaling mechanism for DDR1-mediated SMC migration. In response to type I collagen stimulation, DDR1 is tyrosine phosphorylated within the kinase domain. Src regulates DDR1 phosphorylation and downstream signaling via ERK1/2 and influences cell migration. A proposed model for our studies can be found in Fig. 6. Previous studies in other cell types have suggested that the non-receptor tyrosine kinase Src modulates DDR1 function. Src regulates DDR1 tyrosine phosphorylation in mammary epithelial cells [8] and is involved in shedding of the DDR1 ectodomain in breast carcinoma cells [9]. Our current studies show that Src is associated with DDR1 in vascular SMCs; however, the co-immunoprecipitation experiments suggest that only a small fraction of the pool of DDR1 binds to Src. Nevertheless, inhibition of Src activity reduces the phosphorylation of DDR1. Consequently, this Src/DDR1 association has a great influence on DDR1 tyrosine phosphorylation. We have also shown that DDR1 is required for the collagen-stimulated activation of ERK1/2 in SMCs. By contrast, collagen stimulates p38 signaling even in the absence of DDR1. Furthermore, the Src inhibitor PP2 inhibited the activation of ERK1/2 in SMCs, but did not inhibit p38 activation. This suggests that Src activates ERK1/ 2 pathways downstream of DDR1, whereas activation of p38 likely occurs via integrin signaling and is independent of Src. This is consistent with results from studies that have shown that integrin receptor signaling activates p38, which is important in mediating cell migration and contraction of three-dimensional collagen matrices [23].
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Our results add to a body of literature which reveals that there is substantial variation in DDR1 signaling. For example, in mesangial cells, DDR1 suppresses ERK1/2 activation to maintain cell quiescence [11], while in mammary epithelial cells DDR1 stimulation leads to ERK1/2 activation and cell proliferation [12]. In macrophages, DDR1b promotes activation of p38 [24], ERK1/2, and JNK [13]. DDR1 also signals via JNK to mediate the epithelial to mesenchymal transition of pancreatic cancer cells [14]. The context in which a cell is exposed to the extracellular matrix may also be very important; when DDR1 is overexpressed in Madin–Darby canine kidney cells grown in three-dimensional collagen, migration is inhibited [25]. Future studies are needed to determine whether there are differences in the expression of receptor isoforms or differences in the type or the physical state of collagen that signal via DDR1. In SMCs, migration in response to collagen was critically dependent upon DDR1, as DDR1+/+ cells migrated more than DDR1−/− cells. Migration was also dependent upon Src activity, as PP2 treatment decreased the migration of both DDR1+/+ and DDR1−/− cells. Furthermore, Src inhibition had a greater effect in attenuating the migration of DDR1+/+ SMCs, suggesting that Src is downstream of DDR1 in regulating migration. The MEK inhibitor also inhibited cell migration, although not as strongly as the Src inhibitor, and there was no difference in migration dependent upon DDR1 expression. This suggests that although activation of ERK1/2 in response to collagen stimulation is dependent upon DDR1, ERK1/2 does not mediate DDR1-dependent differences in cell migration. Instead, other factors downstream of Src must be involved, and this will require further study. Taken together, our data indicates that collagen stimulates SMC migration which is mediated through DDR1 activating Src downstream of the receptor. References [1] Adiguzel E, Ahmad PJ, Franco C, Bendeck MP. Collagens in the progression and complications of atherosclerosis. Vasc Med 2009;14: 73–89. [2] Vogel W, Gish GD, Alves F, Pawson T. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol Cell 1997;1:13–23. [3] Ferri N, Carragher NO, Raines EW. Role of discoidin domain receptors 1 and 2 in human smooth muscle cell-mediated collagen remodeling: potential implications in atherosclerosis and lymphangioleiomyomatosis. Am J Pathol 2004;164:1575–85. [4] Franco C, Hou G, Ahmad PJ, Fu EY, Koh L, Vogel WF, et al. Discoidin domain receptor 1 (Ddr1) deletion decreases atherosclerosis by accelerating matrix accumulation and reducing inflammation in low-density lipoprotein receptor deficient mice. Circ Res 2008;102: 1202–11. [5] Hou G, Vogel W, Bendeck MP. The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair. J Clin Invest 2001;107: 727–35. [6] Koo DH, McFadden C, Huang Y, Abdulhussein R, Friese-Hamim M, Vogel WF. Pinpointing phosphotyrosine-dependent interactions downstream of the collagen receptor DDR1. FEBS Lett 2006;580: 15–22.
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