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Gary K Owens – One of the best experts on this subject based on the ideXlab platform.

  • the smooth muscle alpha Actin gene promoter is differentially regulated in smooth muscle versus non smooth muscle cells
    Journal of Biological Chemistry, 1995
    Co-Authors: Richard T Shimizu, Randal S. Blank, Ramiro Jervis, S C Lawrenzsmith, Gary K Owens

    Abstract:

    Abstract To identify potential regulators of smooth muscle cell (SMC) differentiation, we studied the molecular mechanisms that control the tissue-specific transcriptional expression of SM α-Actin, the most abundant protein in fully differentiated SMCs. A construct containing the region from −1 to −125 of the promoter (p125CAT) had high transcriptional activity in SMCs (57-fold > promoterless) and endothelial cells (ECs) (18-fold) but not in skeletal myoblasts or myotubes. Mutation of either of two highly conserved CC(AT-rich)6GG (CArG) motifs at −62 and −112 abolished the activity of p125CAT in SMCs but had no effect in ECs. In contrast, high transcriptional activity in skeletal myotubes, which also express SM α-Actin, required at least 271 base pairs of the promoter (−1 to ≥ −271). Constructs containing 547 base pairs or more of the promoter were transcriptionally active in SMCs and skeletal myotubes but had no activity in skeletal myoblasts or ECs, cell types that do not express SM α-Actin. Electrophoretic mobility shift assays provided evidence for binding of a unique serum response factor-containing complex of factors to the CArG box elements in SMCs. Results indicate that: 1) transcriptional expression of SM α-Actin in SMCs requires the interaction of the CArG boxes with SMC nucleoprotein(s); 2) expression of SM α-Actin in skeletal myotubes requires different cis-elements and trans-factors than in SMCs; and 3) negative-Acting cis-elements are important in restricting transcription in cells that do not express SM α-Actin.

  • elements of the smooth muscle alpha Actin promoter required in cis for transcriptional activation in smooth muscle evidence for cell type specific regulation
    Journal of Biological Chemistry, 1992
    Co-Authors: Randal S. Blank, Tim C. Mcquinn, Maria M. Thompson, Kunio Takeyasu, Robert J Schwartz, Gary K Owens

    Abstract:

    Abstract To assess the role of cis-Acting elements within the smooth muscle alpha-Actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-Actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions -151 and -257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-Actin gene was isolated, and the 5′-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position -214 in the chicken gene and at position -213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-Acting elements than other cell types to transcriptionally regulate this gene.

Kathleen G Morgan – One of the best experts on this subject based on the ideXlab platform.

  • Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator stimulated phosphoprotein vasp
    Biophysical Journal, 2009
    Co-Authors: Francois Ferron, Malgorzata Boczkowska, Philip Graceffa, Cynthia Gallant, Paul C Leavis, Roberto Dominguez, Kathleen G Morgan

    Abstract:

    Our group has shown that alpha agonists and phorbol esters increase net Actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that Actin polymerization is linked to contractility. However, the underlying mechanisms are still largely unknown. Inhibition of Actin filament elongation by cytochalasin-D treatment decreases contractility without changing the level of myosin light chain phosphorylation in this tissue, suggesting that Actin filament elongation processes are necessary for smooth muscle contraction. The enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is associated with Actin filament elongation in non-muscle systems. In this study, we evaluated the possible functions of Ena/VASP in dVSMC. Among Ena/VASP proteins, only VASP is highly expressed in ferret aorta. High resolution 3-D deconvolved fluorescent images of immunostained freshly dissociated aorta cells show that VASP partially colocalizes with both alpha-Actinin and vinculin, markers of dense bodies and dense plaques in dVSMC. Profilin, which is known to associate with monomeric G-Actin and VASP to facilitate Actin filament elongation also colocalizes with both alpha-Actinin and vinculin, potentially identifying both the dense bodies and the dense plaques as hot spots of Actin polymerization. Differential centrifugation and imaging data indicate that VASP may undergo subtle conformational or/and positional changes in response to stimuli. The EVH1 domain of VASP is known to be responsible for targeting VASP to its sites of action. Introduction of an expressed EVH1 domain of Ena/VASP, made as a chimeric protein with the TAT transduction tag, acted as a decoy to inhibit stimulus-induced increases in Actin polymerization. In contrast, introduction of the EVH1 mutant F78S, which does not bind target poly-Pro sequences, had no effect. Thus, VASP may be involved in Actin filament assembly at dense bodies and dense plaques in dVSMC. Support: NIH P01 HL86655.

Christine Chaponnier – One of the best experts on this subject based on the ideXlab platform.

  • the n terminal ac eeed sequence plays a role in alpha smooth muscle Actin incorporation into stress fibers
    Journal of Cell Science, 2005
    Co-Authors: Sophie Clement, Boris Hinz, V B Dugina, Giulio Gabbiani, Christine Chaponnier

    Abstract:

    We have previously shown that the N-terminal sequence AcEEED of alpha-smooth-muscle Actin causes the loss of alpha-smooth-muscle Actin from stress fibers and a decrease in cell contractility when introduced in myofibroblasts as a cell-penetrating fusion peptide. Here, we have investigated the function of this sequence on stress fiber organization in living cells, using enhanced green fluorescent protein (EGFP)-tagged alpha-smooth-muscle Actin. The fusion peptide provokes the gradual disappearance of EGFP fluorescence of alpha-smooth-muscle Actin from stress fibers and the formation of hitherto unknown rod-like structures. In addition to alpha-smooth-muscle Actin, these structures contain cytoplasmic Actins, gelsolin and cofilin but not other major Actin-binding proteins. These rod-like structures are also visible in wild-type fibroblasts during normal cell spreading, suggesting that they represent a physiological step in the organization of alpha-smooth-muscle Actin in stress fibers. Fluorescence-recovery-after-photobleaching experiments suggest that the fusion peptide reduces the dynamics of alpha-smooth-muscle Actin and its incorporation in stress fibers. Here, we propose a new mechanism of how alpha-smooth-muscle Actin is incorporated in stress fibers involving the sequence Ac-EEED.

  • the specific nh2 terminal sequence ac eeed of alpha smooth muscle Actin plays a role in polymerization in vitro and in vivo
    Journal of Cell Biology, 1995
    Co-Authors: Christine Chaponnier, Giulio Gabbiani, M Goethals, Paul A Janmey, F Gabbiani, Joel Vandekerckhove

    Abstract:

    The blocking effect of the NH2-terminal decapeptide of alpha-smooth muscle (SM) Actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-alpha SM-1 (Skalli, O., P. Ropraz, A. Trzeviak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. J. Cell Biol. 103:2787-2796) was compared with that of synthetic peptides modified by changing the acetyl group or by substituting an amino acid in positions 1 to 5. Using immunofluorescence and immunoblotting techniques, anti-alpha SM-1 binding was abolished by the native peptide and by peptides with a substitution in position 5, indicating that AcEEED is the epitope for anti-alpha SM-1. Incubation of anti-alpha SM-1 (or of its Fab fragment) with arterial SM Actin increased polymerization in physiological salt conditions; the antibody binding did not hinder the incorporation of the Actin antibody complex into the filaments. This action was not exerted on skeletal muscle Actin. After microinjection of the alpha-SM Actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM Actin and total Actin showed a selective disappearance of alpha-SM Actin staining, detectable at approximately 30 min. When a control peptide (e.g. alpha-skeletal [SK] Actin NH2-terminal peptide) was microinjected, this was not seen. This effect is compatible with the possibility that the epitopic peptide traps a protein involved in alpha-SM Actin polymerization during the dynamic filament turnover in stress fibers. Whatever the mechanism, this is the first evidence that the NH2 terminus of an Actin isoform plays a role in the regulation of polymerization in vitro and in vivo.