Src modulates contractile vascular smooth muscle function via regulation of focal adhesions

Abstract

Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM. Furthermore, Src inhibition via morpholino knockdown of Src or by the small molecule inhibitor PP2 prevents phenylephrine-induced adhesion protein phosphorylation, markedly slows the tissue's ability to contract, and decreases steady state contractile force amplitude. Significant vasoconstrictor-induced and Src-dependent phosphorylation of Cas pY-165, FAK pY-925, paxillin pY-118, and Erk1/2 were observed. However, increases in FAK 397 phosphorylation were not seen, demonstrating differences between cells in tissue versus migrating, proliferating cells. We show here that Src, in a cause and effect manner, regulates focal adhesion protein function and, consequently, modulates contractility during the action of a vasoconstrictor. These data point to the possibility that vascular focal adhesion proteins may be useful drug discovery targets for novel therapeutic approaches to cardiovascular disease.

Figure 1. Phosphotyrosine screening of aorta samples demonstrates PE-dependent increases and PP2-dependent decreases

(A) Typical blot, phosphotyrosine staining of homogenized samples is shown for samples frozen at slack length, stretched to optimal length (2g stretch), stretched strips exposed to 10⁻5 M PE for 10 min and 10 min 10⁻5 M PE stimulated strips pretreated with 10 mM PP2, a Src inhibitor. (B) Average densitometry of phosphotyrosine staining of bands of indicated molecular mass, normalized to staining for total PKC used as a loading control. n=3. *p<0.05, **p<0.01, ***p<0.001 compared to unstimulated slack strip; +p<0.05, ++p<0.01 compared to PE+PP2 strip.

Figure 2. Tyrosine phosphorylation of FA proteins

The graphs show the densitometric analysis of normalized tyrosine phosphorylation of (A) Cas, (B) paxillin, (C) FAK tyrosine 397, (D) FAK tyrosine 925 and (E) ERK in response to PE. Typical western blots with samples quick frozen at the indicated conditions are shown below the graphs. Note that tyrosine phosphorylation of Cas, paxillin, ERK and FAK tyrosine 925, but not FAK tyrosine 397, are sensitive to the Src inhibitor PP2. n=3–4. ## p<0.01, ### p<0.001 compared to stretch only, *** p<0.001 compared to unstimulated slack, + p<0.05, +++ p<0.001 compared PP2.

Figure 3. Src phosphorylation in response to PE and effect of Src inhibitor PP2 on force

(A) p60 overlaps exactly with Src in a 2-color LiCor blot. (B and C) Src phosphorylation is not detected at either Y416 (B) or at Y529 (C) after stretch or PE. The graphs represent average densitometry data of normalized Src phosphorylation at the indicated sites (n=3–5). (D–F) Src inhibition with PP2 slows the time course and decreases the maximum force production in the presence of PE (n=3–6). (D) Chart recording to illustrate how measurements were made. (E) Time to 50% maximal force production is increased after the addition of PE in the presence of pretreatment with PP2. (F) Maximal amplitude of contraction to PE is decreased by pretreatment with PP2. * p<0.05 compared to PE. (G) Pretreatment with PP2 does not affect myosin light chain phosphorylation.

Figure 4. Effects of Src antisense morpholino knockdown on FA protein / ERK phosphorylation and force

(A) PCRs with specific primers show that at least three members of the Src family, Src, Fgr and Hck, are expressed in dVSM (agarose gel image inverted for optimal display). (B) Morpholino Src family knockdown downregulates Src and Fgr expression but upregulates Hck expression. In each case the lanes are from the same blot but an intervening blank lane was spliced out of the Hck blot. (C) Morpholino Src knockdown decreases tyrosine phosphorylation of FA proteins Cas, FAK and paxillin, as well as ERK phosphorylation. Phosphotyrosine screening demonstrates an inhibition of PE-stimulated increases in phosphotyrosine at 135 kDa (Cas), 125 kDa (FAK) and 75kDa (paxillin). ERK phosphorylation was detected with a phospho-specific ERK antibody. Graph shows average densitometry values (n=3–8). (D) Time to 50% force generation is increased by Src knockdown (graph on left) and PE-stimulated increase in maximal force is inhibited by Src knockdown (graph on right). n=3–8. * p<0.05 compared to control, + p< 0.05 compared to morpholino.

Figure 5. PE causes cellular redistribution of Src and the FA protein Cas

(A.) Deconvolution microscopy (center sections) of Src/Vinculin co-stained unstimulated cell shows adjacency of the 2 proteins in unstimulated cells and an increase in central vesicular-like staining in the presence of PE. (B) Differential centrifugation analysis of Src (left graph) and Cas (right graph) in unstimulated (light gray bars) and PE (dark gray bars) stimulated tissues. Cytosolic, membrane, and cytoskeletal fractions normalized as a percentage of the total (n = 5–6). * p<0.05 compared to resting values.