Transgelin-1 (SM22α) interacts with actin stress fibers and podosomes in smooth muscle cells without using its actin binding site

2018 ◽  
Vol 505 (3) ◽  
pp. 879-884 ◽  
Author(s):  
Tsubasa S. Matsui ◽  
Akihiro Ishikawa ◽  
Shinji Deguchi
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Site ◽  
Muscle Cells ◽  
Stress Fibers ◽  
Actin Binding ◽  
Actin Binding Site ◽  
Actin Stress Fibers

Down-regulation of calponin destabilizes actin cytoskeletal structure in A7r5 cells

2007 ◽  
Vol 85 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Ava C. Dykes ◽  
Gary L. Wright
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Transforming Growth Factor ◽  
Muscle Cells ◽  
Stress Fibers ◽  
Resting Cells ◽  
Tgf Β1 ◽  
Actin Stress Fibers

The effects of changes in the expression levels of h1 calponin (CaP) on actin cytoskeletal organization were studied in control and phorbol-ester-treated A7r5 smooth muscle cells. Protein association and expression in control and stimulated A7r5 smooth muscle cells were evaluated by Western blotting, laser scanning confocal microscopy (LSCM), and fluorescence resonance energy transfer (FRET) microscopy in cells treated with either 2 × 10−6 mol/L TGF-β1 or 2 × 10−5 mol/L PDGF-BB to alter h1 calponin expression. Single immunostained samples showed that CaP and α-actin, localized in fibers in unstimulated control A7r5 smooth muscle cells, were translocated to podosomes following treatment with phorbol-12,13-dibutyrate (PDBu). Confocal colocalization imaging and FRET analysis both indicated substantial association of CaP with α-actin in stress fibers of control cells and in podosomes of PDBu-treated cells. PKCα, which showed evidence of only slight association with CaP in control cells, exhibited markedly increased (293%) association in PDBu-contracted cells. Platelet-derived growth factor (PDGF)-BB down-regulated CaP to non-detectable levels, whereas transforming growth factor (TGF)-β1 up-regulated (424%) the expression of CaP without affecting the levels of α-actin or PKCα. PDGF-BB resulted in a significant loss in α-actin stress fibers (–47%) and reduced podosome formation (–69%). By comparison, TGF-β1 had no effect on stress fibers in control cells but also reduced (–70%) podosome formation. The results suggest that CaP could play a major role in the stabilization of actin stress fibers in resting cells and may contribute to podosome formation in PDBu-treated cells.

Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells

2003 ◽  
Vol 285 (4) ◽  
pp. L829-L837 ◽  
Author(s):  
Gabor Horvath ◽  
Zoltan Sutto ◽  
Aliza Torbati ◽  
Gregory E. Conner ◽  
Matthias Salathe ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Binding Site ◽  
Membrane Binding ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Extraneuronal Monoamine Transporter ◽  
Arterial Smooth Muscle Cells ◽  
Specific Membrane

Inhaled glucocorticosteroids (GSs) cause acute, α1-adrenoreceptor (AR)-mediated bronchial vasoconstriction. After release from sympathetic nerves, norepinephrine (NE) must be taken up into cells for deactivation by intracellular enzymes. Because postsynaptic cellular NE uptake is steroid sensitive, GSs could increase NE concentrations at α1-AR, causing vasoconstriction. We therefore evaluated mRNA expression of different NE transporters in human bronchial arterial smooth muscle and pharmacologically characterized NE uptake into these cells. RT-PCR demonstrated mRNA expression of the extraneuronal monoamine transporter (EMT) and organic cation transporter 1 (OCT-1). Fluorometric uptake assay showed time (within minutes)- and concentration-dependent NE uptake by freshly isolated bronchial arterial smooth muscle cells (SMC) with an estimated Km of 240 μM. Corticosterone and O-methylisoprenaline (1 μM each), but not desipramine, inhibited NE uptake, a profile indicative of NE uptake by EMT, but not OCT-1. Budesonide and methylprednisolone inhibited uptake with IC50 values of 0.9 and 5.6 μM, respectively. Corticosterone's action was reversible and not sensitive to RU-486 (GS receptor antagonist), actinomycin D (transcription inhibitor), or cycloheximide (protein synthesis inhibitor). Corticosterone made membrane impermeant by coupling to BSA also blocked NE uptake. Immunocytochemistry indicated a specific membrane binding site for corticosterone on bronchial arterial SMC. These data demonstrate that although human bronchial arterial SMC express OCT-1 and EMT, EMT is the predominant plasma membrane transporter for NE uptake. This process can be inhibited by GSs, likely via a specific membrane binding site. This nongenomic GS action (increasing NE concentrations at α1-AR) could explain acute bronchial vasoconstriction caused by inhaled GSs.

Abstract 273: P2Y2 Receptor-Mediated Lymphotoxin-α Secretion Regulates ICAM-1 Expression in Smooth Muscle Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Cheikh Seye ◽  
Wilbert Derbigny ◽  
Shaomin Qian
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Protein Transport ◽  
Brefeldin A ◽  
Muscle Cells ◽  
Secretory Vesicle ◽  
Actin Binding ◽  
Filamin A ◽  
Artery Disease ◽  
Lymphotoxin Α

Rationale: Single nucleotide polymorphism (SNP) in the LGASL2 galectin-2 (Gal-2) gene leads to altered secretion of lymphotoxin-α (LT-α) and is associated with coronary artery disease. Objective:Our aim was to determine whether factors other than genetic variations in LGASL2 regulate LT-α release and to define the role of this pro-inflammatory in vascular smooth muscle cells (SMC). Methods and results: The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y 2 nucleotide receptor (P2Y 2 R) agonist UTP stimulates a strong and sustained release of LTA from wild-type but not P2Y 2 R -/- SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi apparatus, suggesting that P2Y 2 R/filamin-mediated secretion of LTA is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y 2 R deficient in LTA secretion. Conclusion: These data suggest that P2Y 2 R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.

scholarly journals Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

2013 ◽  
Vol 305 (8) ◽  
pp. C829-C845 ◽  
Author(s):  
Hanaa K. B. Motawea ◽  
Selvi C. Jeyaraj ◽  
Ali H. Eid ◽  
Srabani Mitra ◽  
Nicholas T. Unger ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Smooth Muscle Cells ◽  
Blood Vessels ◽  
Muscle Cells ◽  
Small Gtpase ◽  
Actin Binding ◽  
Surface Translocation

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

scholarly journals CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

1997 ◽  
Vol 139 (1) ◽  
pp. 157-168 ◽  
Author(s):  
Pascal Pomiès ◽  
Heather A. Louis ◽  
Mary C. Beckerle
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Actin Cytoskeleton ◽  
Indirect Immunofluorescence ◽  
Muscle Cells ◽  
Muscle Differentiation ◽  
Full Length ◽  
Stress Fibers ◽  
Lim Domain

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is α-actinin. We have shown that the CRP1–α-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1–α-actinin interaction is 1.8 ± 0.3 μM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and α-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that α-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin–binding domain of α-actinin. In reciprocal mapping studies, we showed that α-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the α-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the α-actinin–binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with α-actinin may be critical for its role in muscle differentiation.

Sign in / Sign up

Export Citation Format

Share Document