The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility
"> Figure 1
<p>PAR2–AP- and TGF-β1-driven random cell migration are dependent on ERK activation. Panc1 cells (left-hand graph) and Colo357 cells (right-hand graph) were subjected to a cell migration assay (chemokinesis setup) in the presence of vehicle (0.1% dimethylsulfoxide, DMSO) or 20 µM U0126 and either PAR2–AP (15 µM 2-furoyl-LIGRLO-NH<sub>2</sub> (2f-LI), left-hand graph) or TGF-β1 (5 ng/mL, right-hand graph). In the left graph, differences are significant (<span class="html-italic">p</span> < 0.05, unpaired Student’s <span class="html-italic">t</span>-test) between vehicle + PAR2–AP treated cells (blue curve, tracing B) and U0126 + PAR2–AP treated cells (magenta curve, tracing D) at 4:00 and all later time points, and between vehicle treated cells (red curve, tracing A) and U0126 treated cells (green curve, tracing C) at 6:00 and all later time points. In the right-hand graph, differences are significant between vehicle + TGF-β1 treated cells (blue curve, tracing B) and U0126 + TGF-β1 treated cells (magenta curve, tracing D) at 12:00 and all later time points. For a color-independent identification of the curves, see letters to the right of each graph. In each graph, data are shown from one representative experiment; three experiments were performed in total.</p> "> Figure 2
<p>Kinetics of ERK activation in response to PAR2–AP or TGF-β1 in various cell types. (<b>a</b>) Panc1 (left) and HaCaT (right) cells were stimulated with 2f-LI (15 µM) for 2–240 min and subsequently analyzed by phospho-immunoblotting for phospho-ERK1/2 (p-ERK1/2). Following removal of the p-ERK1/2 antibody, the blot was incubated with antibodies to total ERK1/2 and to HSP90 as a loading control; (<b>b</b>) Panc1 and Colo357 cells were stimulated with TGF-β1 (5 ng/mL) for 1–12 h and subjected to immunoblot analysis for p-ERK1/2, ERK, and HSP90 as described in (<b>a</b>). The graphs below the immunoblots show the results from the densitometric analysis of four independent experiments (mean ± SD). The asterisks indicate significant differences relative to the untreated control, <span class="html-italic">p</span> < 0.05.</p> "> Figure 3
<p>ERK1/2 but not SMAD3 activation in response to TGF-β1 was blocked by the MEK inhibitor U0126. The indicated cell lines were grown to confluence, starved for 24 h in medium containing 0.1% bovine serum albumin, and treated for the indicated times with TGF-β1 (T, 5 ng/mL) in the absence or presence of either vehicle (V, 0.2% DMSO), the MEK inhibitor U0126 (U, 20 µM), or the Rac1 inhibitor NSC23766 (N, 200 µM) as negative control. Cells were subjected to immunoblotting for p-ERK1/2, ERK1/2, p-SMAD3C, and SMAD3 and for HSP90 to control for equal loading. The total forms of ERK1/2 and SMAD3 were not different between the various time points and treatments. The functionality of U0126 was confirmed by its ability to block ERK1/2 activation after a 5 min challenge of cells with EGF (E, 10 ng/mL).</p> "> Figure 4
<p>Both PAR2–AP- and TGF-β1-induced ERK activation are dependent on PAR2 protein expression. (<b>a</b>) Panc1 cells were transiently transfected with 50 nM of either control (Co) siRNA or siRNA specific to PAR2 (PAR2). Following stimulation with PAR2–AP (P2-AP), PAR1–AP (P1-AP) for 5 min or TGF-β (T) for 1 h, cells were subjected to immunoblotting for p-ERK1/2 and ERK1/2, and for HSP90 as a loading control. The graph below the blot shows densitometric data (mean ± SD) of underexposed bands derived from three parallel wells. One representative experiment is shown out of three performed in total. Asterisks indicate significance <span class="html-italic">p</span> < 0.05; (<b>b</b>) HaCaT cells were transfected with 50 nM of either Co siRNA or PAR2 siRNA, stimulated for the indicated times with TGF-β1, and processed for immunoblotting of p-ERK1/2 and ERK1/2. The graphs below the blots show results from densitometry-based quantification of three experiments, mean ± SD. The asterisk indicates significance <span class="html-italic">p</span> < 0.05.</p> "> Figure 5
<p>GB88 increases basal and TGF-β1-induced ERK activation and cell migration. (<b>a</b>) Panc1 cells were serum starved (1% fetal bovine serum, FBS) for 20 h prior to treatment for the indicated times with vehicle (V) or GB88 at concentrations (conc.) of either 10 µM or 100 µM. Crude cellular lysates were immunoblotted for p-ERK1/2, ERK1/2, and HSP90, and underexposed replicas subjected to densitometric analysis. Data in the graph represent the mean ± SD of three bands derived from cells from three parallel wells. One representative experiment is shown out of three performed in total. Asterisks indicate significance <span class="html-italic">p</span> < 0.05; (<b>b</b>) Panc1 cells were subjected to real-time cell migration assays in the presence of vehicle (0.1% DMSO) and either 10 µM of GB88 (left-hand graph) or GB110 (right-hand graph). In the left-hand graph, differences are significant (<span class="html-italic">p</span> < 0.05, unpaired Student’s <span class="html-italic">t</span>-test) between vehicle + TGF-β1 treated cells (blue curve, tracing B) and GB88 + TGF-β1 treated cells (magenta curve, tracing D) at 8:00 and all later time points. In the right-hand graph, differences are significant between vehicle + TGF-β1 treated cells (blue curve, tracing B) and GB110 + TGF-β1 treated cells (magenta curve, tracing D) at 4:00 and all later time points. In each graph, data are shown from one representative experiment out of three experiments performed in total.</p> "> Figure 6
<p>PAR2 and activin receptor-like kinase 5 (ALK5) can be co-immunoprecipitated. Panc1 cells were transfected with empty vector, PAR2-Myc-DKK, or ALK5-HA, alone or in combination, as indicated. Two days later, total cell lysates (20 µg each) were sequentially immunoblotted (IB) with IgG<sub>2a</sub> as negative control (left blot), and with anti-Myc tag (middle blot) and anti-ALK5 antibodies (right blot) for detection of transfected PAR2 and transfected (and endogenous) ALK5 protein, respectively. The appearance of bands for endogenous ALK5 indicates equal protein loading. Subsequently, anti-Myc or anti-His microbead-based IP was used on ~1 mg of lysate (duplicate samples) to precipitate PAR2–Myc–DKK along with associated proteins followed by immunoblotting for ALK5 (upper panel) and anti-Myc tag (lower panel). Numbers next to the molecular weight marker (M) lanes indicate the molecular mass (in kDa). Note that PAR2–Myc–DKK migrates as a complex of diffuse bands between ~100 and >250 kDa because of heavy glycosylation [<a href="#B35-ijms-18-02776" class="html-bibr">35</a>,<a href="#B36-ijms-18-02776" class="html-bibr">36</a>]. Data shown are from a representative experiment out of five experiments performed in total.</p> ">
Abstract
:1. Introduction
2. Results
2.1. ERK Activation Is Required for Both PAR2–AP- and TGF-β1-Mediated Cell Migration
2.2. ERK Is Activated by PAR2–AP and TGF-β1 with Different Kinetics
2.3. ERK but Not SMAD Activation in Response to TGF-β1 Was Blocked by the MEK Inhibitor U0126
2.4. Both PAR2-AP- and TGF-β1-Induced ERK Activation are Dependent on PAR2 Protein Expression
2.5. Treatment with High Concentrations of GB88 Increases Basal and TGF-β1-Induced ERK Activation and Cell Migration
2.6. PAR2 and ALK5 Can Be Co-Immunoprecipitated
3. Discussion
4. Materials and Methods
4.1. Reagents and Antibodies
4.2. Cell Culture
4.3. Transient Transfection of siRNAs
4.4. Immunoblot Analysis and Co-Immunoprecipitation
4.5. Migration Assays
4.6. Statistical Analysis
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
ALK5 | Activin receptor-like kinase 5 |
AP | Agonistic peptide |
ERK | Extracellular signal-regulated kinase |
PAR2 | Proteinase-activated receptor 2 |
TGF-β | Transforming growth factor-β |
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Ungefroren, H.; Witte, D.; Fiedler, C.; Gädeken, T.; Kaufmann, R.; Lehnert, H.; Gieseler, F.; Rauch, B.H. The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility. Int. J. Mol. Sci. 2017, 18, 2776. https://doi.org/10.3390/ijms18122776
Ungefroren H, Witte D, Fiedler C, Gädeken T, Kaufmann R, Lehnert H, Gieseler F, Rauch BH. The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility. International Journal of Molecular Sciences. 2017; 18(12):2776. https://doi.org/10.3390/ijms18122776
Chicago/Turabian StyleUngefroren, Hendrik, David Witte, Christian Fiedler, Thomas Gädeken, Roland Kaufmann, Hendrik Lehnert, Frank Gieseler, and Bernhard H. Rauch. 2017. "The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility" International Journal of Molecular Sciences 18, no. 12: 2776. https://doi.org/10.3390/ijms18122776