Susanne Vetterkind

Smooth muscle signalling pathways in health and disease

•  Introduction •  Mechanisms that regulate LC20 phosphorylation ‐  Regulation of myosin phosphatase ‐  CaMKII •  Mechanisms that regulate the access of myosin to actin ‐  Caldesmon ‐  bCaP function in regulation of PKC and ERK signalling •  Mechanisms that regulate cytoskeletal remodelling •  Conclusions

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

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 tissues 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. J. Cell. Physiol. 227: 3585–3592, 2012.

The focal adhesion: a regulated component of aortic stiffness.

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.

Par-4: A New Activator of Myosin Phosphatase

We show here for the first time that the pro-apoptotic protein Par-4 binds to and activates myosin phosphatase (MP). During agonist stimulation, Par-4 facilitates ZIPK targeting and inhibitory phosphorylation of MP, however, phosphorylation of Par-4 is required for MP inhibition. Our model presents Par-4 as an amplifier of the MP activity range.

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK.

Par‐4 (prostate apoptosis response 4) is a pro‐apoptotic protein and tumour suppressor that was originally identified as a gene product up‐regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par‐4 is expressed and co‐localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF‐2α stimulation, is inhibited by the presence of a cell permeant Par‐4 decoy peptide. The same decoy peptide also significantly inhibits PGF‐2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par‐4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par‐4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par‐4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.

Hierarchical scaffolding of an ERK1/2 activation pathway

BackgroundScaffold proteins modulate cellular signaling by facilitating assembly of specific signaling pathways. However, there is at present little information if and how scaffold proteins functionally interact with each other.ResultsHere, we show that two scaffold proteins, caveolin-1 and IQGAP1, are required for phosphorylation of the actin associated pool of extracellular signal regulated kinase 1 and 2 (ERK1/2) in response to protein kinase C activation. We show by immunofluorescence and proximity ligation assays, that IQGAP1 tethers ERK1/2 to actin filaments. Moreover, siRNA experiments demonstrate that IQGAP1 is required for activation of actin-bound ERK1/2. Caveolin-1 is also necessary for phosphorylation of actin-bound ERK1/2 in response to protein kinase C, but is dispensible for ERK1/2 association with actin. Simultaneous knock down of caveolin-1 and IQGAP1 decreases total phorbol ester-induced ERK1/2 phosphorylation to the same degree as single knock down of either caveolin-1 or IQGAP1, indicating that caveolin-1 and IQGAP1 operate in the same ERK activation pathway. We further show that caveolin-1 knock down, but not IQGAP1 knock down, reduces C-Raf phosphorylation in response to phorbol ester stimulation.ConclusionsBased on our data, we suggest that caveolin-1 and IQGAP1 assemble distinct signaling modules, which are then linked in a hierarchical arrangement to generate a functional ERK1/2 activation pathway.

Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle.

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin- and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.

Stimulus-Specific Activation and Actin Dependency of Distinct, Spatially Separated ERK1/2 Fractions in A7r5 Smooth Muscle Cells

A proliferative response of smooth muscle cells to activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) has been linked to cardiovascular disease. In fully differentiated smooth muscle, however, ERK1/2 activation can also regulate contraction. Here, we use A7r5 smooth muscle cells, stimulated with 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) to induce cytoskeletal remodeling or fetal calf serum (FCS) to induce proliferation, to identify factors that determine the outcomes of ERK1/2 activation in smooth muscle. Knock down experiments, immunoprecipitation and proximity ligation assays show that the ERK1/2 scaffold caveolin-1 mediates ERK1/2 activation in response to DPBA, but not FCS, and that ERK1/2 is released from caveolin-1 upon DPBA, but not FCS, stimulation. Conversely, ERK1/2 associated with the actin cytoskeleton is significantly reduced after FCS, but not DPBA stimulation, as determined by Triton X fractionation. Furthermore, cytochalasin treatment inhibits DPBA, but not FCS-induced ERK1/2 phosphorylation, indicating that the actin cytoskeleton is not only a target but also is required for ERK1/2 activation. Our results show that (1) at least two ERK1/2 fractions are regulated separately by specific stimuli, and that (2) the association of ERK1/2 with the actin cytoskeleton regulates the outcome of ERK1/2 signaling.

A novel mechanism of ERK1/2 regulation in smooth muscle involving acetylation of the ERK1/2 scaffold IQGAP1

Ceramide, a bioactive lipid and signaling molecule associated with cardiovascular disease, is known to activate extracellular signal regulated kinases 1 and 2 (ERK1/2). Here, we determined that the effect of ceramide on ERK1/2 is mediated by ceramide signaling on an ERK scaffold protein, IQ motif containing GTPase activating protein 1 (IQGAP1). Experiments were performed with aortic smooth muscle cells using inhibitor screening, small interfering RNA (siRNA), immunoprecipitation (IP), immunoblots and bioinformatics. We report here that C6 ceramide increases serum-stimulated ERK1/2 activation in a manner dependent on the ERK1/2 scaffold IQGAP1. C6 ceramide increases IQGAP1 protein levels by preventing its cleavage. Bioinformatic analysis of the IQGAP1 amino acid sequence revealed potential cleavage sites for proteases of the proprotein convertase family that match the cleavage products. These potential cleavage sites overlap with known motifs for lysine acetylation. Deacetylase inhibitor treatment increased IQGAP1 acetylation and reduced IQGAP1 cleavage. These data are consistent with a model in which IQGAP1 cleavage is regulated by acetylation of the cleavage sites. Activation of ERK1/2 by ceramide, known to increase lysine acetylation, appears to be mediated by acetylation-dependent stabilization of IQGAP1. This novel mechanism could open new possibilities for therapeutic intervention in cardiovascular diseases.

Chapter 87 – Regulation of Smooth Muscle Contraction

Tight regulation of smooth muscle contraction is of tremendous importance for the proper function of organ systems of the body, including circulatory, respiratory, genito-urinary, and digestive systems. Moreover, because of its central role in regulation of blood pressure, smooth muscle contractility indirectly affects virtually all organs of the body. It is therefore not surprising that smooth muscle contraction is controlled and modified at many levels. Electrical, chemical, and mechanical stimulation can trigger smooth muscle contraction. At the molecular level, stimulation is transmitted through various regulatory pathways that aim at only two major targets: actin and myosin. Contractility is modulated by both short-term (e.g. post-translational modification) and long-term (e.g. variations in gene expression) regulatory mechanisms. Scaffold proteins organize the complex network of signaling pathways in space and time.