The cytoskeletal fraction of ERK1/2 is mobilized upon FCS, but not DPBA, stimulation.

<p>(A) A7r5 cells were treated with either DPBA or FCS and then subjected to subcellular fractionation into cytoskeletal and soluble fractions by Triton X-100 extraction. ERK1/2 subcellular distribution was analyzed by western blotting and densitometry. The graph shows the percentage of total ERK1/2 found in the caveolar and non-caveolar (cytoskeletal) TX-insoluble fractions at late time points of stimulation (30 minutes and 60 minutes). Statistical significance was tested by two-tailed Student's T-Tests (non-caveolar insoluble fraction: *p<0.05 DPBA vs. FCS, **p<0.01 unstimulated vs. FCS; caveolar insoluble fraction: ^#p<0.05 DPBA vs. FCS). (B) A7r5 cells were grown on coverslips were subjected to TX extraction before fixing and staining for phospho-ERK1/2. Please note that filamentous phospho-ERK1/2 staining after DPBA stimulation is TX resistant, while the nuclear and diffuse perinuclear phospho-ERK1/2 staining after FCS stimulation is not. Scale bar, 20 µm. (C) A7r5 cells, pretreated with Cytochalasin D for one hour or control treated (solvent only), were stimulated with either FCS or DPBA, or left unstimulated, for another hour. Lysates were subjected to western blotting with a total ERK1/2 antibody and a phospho-ERK1/2 antibody. The graph shows phospho-ERK1/2 relative to total ERK1/2. Statistical significance was tested by a paired two-tailed Student's T-Test (**p<0.01 Cyto D vs. control, #p<0.05 DPBA vs. FCS, n.s. = not significant).</p

Knock down of caveolin-1 interferes with DPBA induced ERK1/2 phosphorylation.

<p>A7r5 cells were transfected with caveolin-1 siRNA, caveolin-2 siRNA as control or left untransfected. Experiments were performed five days after transfection. (A) Western blot analysis and statistical analysis of densitometry analysis show efficient knock down of caveolin-1. Statistical significance was tested by two-tailed Student's T-Tests (***p<0.001 siRNA vs. control, ^###p<0.001 siRNA vs. untransfected). (B) Five days after siRNA transfection, cells were stimulated with either DPBA or FCS, or left untreated. Lysates were analyzed for ERK1/2 phosphorylation by western blotting and densitometry. The graph shows that after caveolin-1 knock down, the rate of ERK1/2 phosphorylation is significantly reduced in response to DPBA, but not FCS. Statistical significance was tested by two-tailed Student's T-Tests (**p<0.01 siRNA vs. control, ^##p<0.01 DPBA vs. FCS).</p

A caveolar fraction of ERK1/2 is phosphorylated and mobilized in a stimulus-specific manner.

<p>(A) A7r5 cells grown on coverslips were stimulated with DPBA or FCS, or left unstimulated, and then fixed and stained with a caveolin-1 antibody and either an ERK1/2 or phospho-ERK1/2 antibody. Cells were processed for proximity ligation assays (Olink) according to the manufacturer's protocol and then analyzed for signal dots indicative of close proximity. (B and C) A7r5 cells were treated with either DPBA or FCS for the indicated times. Lysates were then subjected to immunoprecipitation with an anti-caveolin-1 antibody. Immunoprecipitates were analyzed for co-immunoprecipitation of (B) phospho-ERK1/2 and (C) total ERK1/2 by western blotting and densitometry. Please note that interaction of caveolin-1 with phospho-ERK1/2 is increased at early time points after DPBA, but not FCS stimulation, whereas interaction with total ERK1/2 is reduced at later time points of DPBA, but not FCS stimulation. Statistical significance was tested by two-tailed Student's T-Tests (*p<0.05, ***p<0.001 DPBA vs. unstimulated; ^#p<0.05, ^##p<0.01, ^###p<0.001 DPBA vs. FCS; ⁺p<0.05 FCS vs. unstimulated).</p

Stimulus-specific cytoskeletal localization of phospho-ERK1/2 depends on caveolin-1.

<p>A7r5 cells grown on coverslips were transfected with caveolin-1 siRNA and control siRNA. Five days after transfection, cells were stimulated with either DPBA or FCS for 60 minutes, or left untreated. After stimulation, cells were fixed and stained for caveolin-1 (a, e, I, m), phospho-ERK1/2 (b, f, j, n) and DAPI (c, g, k, o); merged images are shown in panels d, h, l, p. Please note that in the caveolin-1 knock down cells, the filamentous phospho-ERK1/2 staining seen after DPBA stimulation is absent in caveolin-1 knock down cells (arrows). Scale bar, 20 µm.</p

ERK1/2 activation has stimulus-specific effects on the A7r5 cytoskeleton.

<p> (A) ERK1/2 is phosphorylated in response to both, DPBA and FCS. A7r5 cells stimulated with either DPBA or FCS for the indicated time points were processed for western blot analysis of total ERK1/2 and phospo-ERK1/2. After densitometry analysis, no significant differences in ERK1/2 phosphorylation (normalized to total ERK1/2) were detected. (B) A7r5 cells were pre-treated with the MEK inhibitor U0126 for 60 minutes, then stimulated with DPBA or FCS, or left untreated, for additional 60 minutes before preparation of cell lysates. Lysates were analyzed by western blotting and densitometry. Please note that in the presence of the MEK inhibitor, caldesmon phosphorylation in response to DPBA is completely blocked, whereas caldesmon phosphorylation (normalized to tubulin) in response to FCS is reduced by only about 30%. Statistical significance was tested by a two-tailed Student's T-Test (**p<0.01 U0126 vs. control, ^##p<0.01 DPBA vs. FCS). (C) A7r5 cells grown on coverslips were treated as indicated for 60 minutes, then fixed and stained for fluorescence microscopy with phalloidin to visualize actin filaments. Please note the cytoskeletal rearrangements and the appearance of podosomes in the DPBA treated cells (arrows). Scale bar, 20 µm.</p

Model: Spatially distinct ERK1/2 subfractions are activated in a stimulus-specific manner.

<p>Upon FCS stimulation, a cytoskeletal ERK1/2 fraction is activated and released into a soluble ERK1/2 fraction (magenta arrow), enabling nuclear ERK1/2 signaling, whereas the caveolar ERK1/2 fraction is not activated in response to FCS. Phorbol ester stimulation, in contrast, leads to phosphorylation of both, caveolar and cytoskeletal ERK1/2 fractions. Caveolar ERK1/2 is released upon activation (green arrows), while cytoskeletal ERK1/2 is retained at actin filaments. The double headed arrow indicates a functional link between the caveolar and cytoskeletal ERK1/2 fractions.</p

Model.

<p>(A) Tension-induced, Src- and FAK-mediated growth and remodeling of dynamic focal adhesions in aortic VSMCs leads to cell-matrix adhesion strengthening (cortical stiffening) in response to contractile stimulus. This strengthening is required for adequate force and stiffness transmission from the VSMC to the blood vessel wall. (B) Inhibition of Src with PP2 or FAK with FI-14 inhibits FA dynamics and growth, preventing reinforcement of the cell-matrix linkage. As a result, forces and stiffness generated by the activated VSMC cannot propagate efficiently to the tissue. (C) Inhibition of MLCK with ML-9 reduces contractile force and, as a result, lessens reinforcement of the cell-matrix linkage. Consequently, force and stiffness development in the aortic wall are reduced.</p

Aortic tissue stiffening during contractile stimulation is decreased by inhibition of Src, FAK, or MLCK.

<p>(A) Tissue stiffness <i>E</i> measured <i>in vitro</i> during PE-induced contraction at optimum length <i>L</i>_O with small-amplitude (1%), high frequency (40Hz) sinusoidal stretches Δ<i>L</i>. Box height and width for magnified traces: 0.5 mN, 5 µm, and 0.02 s. <i>Upper Inset</i>: Stiffness calculation. <i>Lower Inset</i>: Fluorescent micrograph of aortic ring in cross-section, used to determine ring thickness for calculation of cross-sectional area <i>A</i>. Green: autofluorescent elastic laminae. Blue: cell nuclei. Scale bar, 100 µm. (B) PE-induced stress is significantly lower when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation and contraction to vascular stiffness (n = 4). PE-induced stress is also significantly reduced when pre-treated with Src inhibitor PP2 and FAK inhibitor 14. (C) PE-induced stiffening is significantly reduced when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation to aortic stiffness. Stiffening is also significantly lower when pre-treated with Src inhibitor PP2 and FAK inhibitor 14, indicating a role for Src, FAK, and FA proteins in aortic stiffness. n = 10 untreated, 6 ML-9, 4 PP2, 5 FI-14 rings. *p<0.05,**p<0.01, ***p<0.001, unpaired, two-tailed Student’s t-test.</p

PE induces FAK and Src-mediated tyrosine phosphorylation of FA proteins in dVSMCs.

<p>(A) Typical blot, phosphotyrosine screening of mouse aorta tissue homogenates. PE increases tyrosine phosphorylation, and pre-treatment with Src inhibitor PP2 decreases tyrosine phosphorylation. (B) Mean densitometry of phosphotyrosine bands indicated. (C) Phospho-FAK Y925 increases in response to PE in a PP2-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). (D-E) Phospho-CAS Y165 and phospho-paxillin Y118 increase in response to PE in a FI-14-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). The brightness of the representative bands in Insets C-E has been uniformly altered for visual display; however, unaltered images were used for densitometry quantitation. *p<0.05, **p<0.01 vs. control, +p<0.05, ++p<0.01, +++p<0.001 vs. PP2+PE or FI-14+PE, unpaired, two-tailed Student’s t-test.</p

LPA stimulation increases FA size in a PP2-sensitive manner.

<p>Top: Deconvolution microscopy of FAs. Representative FAs are shown in an expanded view in the insets. All FAs in each cell, not just those featured in the insets, were analyzed to determine the mean FA area. Scale bars: top, 20 µm; bottom, 5 µm. Bottom: Mean FA area is significantly greater in cells stimulated with LPA (n = 82 cells). ***p<0.001, unpaired, two-tailed Student’s t-test.</p