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1.
J Biol Chem ; 287(53): 44761-71, 2012 Dec 28.
Article in English | MEDLINE | ID: mdl-23148210

ABSTRACT

MicroRNA-210 (miR-210) induction is a virtually constant feature of the hypoxic response in both normal and transformed cells, regulating several key aspects of cardiovascular diseases and cancer. We found that miR-210 was induced in normoxic myoblasts upon myogenic differentiation both in vitro and in vivo. miR-210 transcription was activated in an hypoxia-inducible factor 1-α (Hif1a)-dependent manner, and chromatin immunoprecipitation experiments show that Hif1a bound to the miR-210 promoter only in differentiated myotubes. Accordingly, luciferase reporter assays demonstrated the functional relevance of the Hif1a binding site for miR-210 promoter activation in differentiating myoblasts. To investigate the functional relevance of increased miR-210 levels in differentiated myofibers, we blocked miR-210 with complementary locked nucleic acid oligonucleotides (anti-miR-210). We found that C2C12 myoblast cell line differentiation was largely unaffected by anti-miR-210. Likewise, miR-210 inhibition did not affect skeletal muscle regeneration following cardiotoxin damage. However, we found that miR-210 blockade greatly increased myotube sensitivity to oxidative stress and mitochondrial dysfunction. In conclusion, miR-210 is induced in normoxic myofibers, playing a cytoprotective role.


Subject(s)
Cell Differentiation , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , MicroRNAs/genetics , Myoblasts/cytology , Myoblasts/metabolism , Oxygen/metabolism , Animals , Base Sequence , Cell Line , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice , MicroRNAs/metabolism , Molecular Sequence Data , Promoter Regions, Genetic
2.
Exp Cell Res ; 315(15): 2581-92, 2009 Sep 10.
Article in English | MEDLINE | ID: mdl-19540230

ABSTRACT

Podosome formation in vascular smooth muscle cells is characterized by the recruitment of AFAP-110, p190RhoGAP, and cortactin, which have specific roles in Src activation, local down-regulation of RhoA activity, and actin polymerization, respectively. However, the molecular mechanism that underlies their specific recruitment to podosomes remains unknown. The scaffold protein Tks5 is localized to podosomes in Src-transformed fibroblasts and in smooth muscle cells, and may serve as a specific recruiting adapter for various components during podosome formation. We show here that induced mislocalization of Tks5 to the surface of mitochondria leads to a major subcellular redistribution of AFAP-110, p190RhoGAP, and cortactin, and to inhibition of podosome formation. Analysis of a series of similarly mistargeted deletion mutants of Tks5 indicates that the fifth SH3 domain is essential for this recruitment. A Tks5 mutant lacking the PX domain also inhibits podosome formation and induces the redistribution of AFAP-110, p190RhoGAP, and cortactin to the perinuclear area. By expressing a catalytically inactive point mutant and by siRNA-mediated expression knock-down we also provide evidence that p190RhoGAP is required for podosome formation. Together our findings demonstrate that Tks5 plays a central role in the recruitment of AFAP-110, p190RhoGAP, and cortactin to drive podosome formation.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Cell Surface Extensions/metabolism , Cortactin/metabolism , Microfilament Proteins/metabolism , Myocytes, Smooth Muscle , Phosphoproteins/metabolism , Repressor Proteins/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Animals , Cell Surface Extensions/ultrastructure , Cortactin/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Microfilament Proteins/genetics , Mitochondria/metabolism , Mitochondria/ultrastructure , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Phosphoproteins/genetics , Point Mutation , Protein Structure, Tertiary , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics
3.
PLoS One ; 3(11): e3638, 2008.
Article in English | MEDLINE | ID: mdl-18982058

ABSTRACT

BACKGROUND: Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion. PRINCIPAL FINDINGS: We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with beta-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation. CONCLUSIONS: We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.


Subject(s)
Cell Adhesion/physiology , Cell Surface Extensions/metabolism , Dystroglycans/metabolism , Myoblasts/cytology , Phosphoproteins/metabolism , src-Family Kinases/metabolism , Actins/metabolism , Animals , Biomarkers/metabolism , Cell Surface Extensions/ultrastructure , Cells, Cultured , Cortactin/metabolism , Dystroglycans/genetics , Humans , Mice , Myoblasts/physiology , Phosphate-Binding Proteins , Phosphoproteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , src Homology Domains , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/genetics
4.
Cell Motil Cytoskeleton ; 65(8): 614-25, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18553359

ABSTRACT

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.


Subject(s)
Myosins/metabolism , Plectin/metabolism , Pseudopodia/metabolism , Actins/metabolism , Animals , Blotting, Western , Cell Line , Cell Movement/drug effects , Cell Movement/physiology , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Focal Adhesions/metabolism , Heterocyclic Compounds, 4 or More Rings/pharmacology , Intermediate Filaments/metabolism , Microscopy, Fluorescence , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Myosins/physiology , Phorbol 12,13-Dibutyrate/pharmacology , Plectin/genetics , Pseudopodia/drug effects , Pseudopodia/physiology , RNA Interference , Rats , Vimentin/metabolism
5.
Eur J Cell Biol ; 85(3-4): 183-9, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16546560

ABSTRACT

Activation of protein kinase C by phorbol esters triggers the remodelling of the actin cytoskeleton and the formation of podosomes in smooth muscle cells (SMCs). Regional control of actin dynamics at specialised microdomains results in a local reduction in contractile forces. The molecular basis for this local inhibition of contractility includes the clustering of cortactin during podosome formation (which precedes the rapid, local dispersion of myosin, tropomyosin and h1 calponin), and the specific recruitment of 110-kDa actin filament-associated protein (AFAP-110) and 190-kDa Rho-specific GTPase-activating protein (p190RhoGAP) to the microdomains. Podosome formation also correlates with cell polarisation, the induction of cell motility, and local degradation of the extracellular matrix. These findings may provide explanations for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis, and support the concept that matrix degradation and the concomitant engagement of the molecular machinery initiating actin-based cell motility drive tissue invasion in smooth muscle.


Subject(s)
Actin Cytoskeleton/ultrastructure , Extracellular Matrix/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/ultrastructure , Actin Cytoskeleton/metabolism , Animals , Cell Differentiation/physiology , Focal Adhesions/metabolism , Focal Adhesions/ultrastructure , Humans , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Signal Transduction/physiology , Substrate Specificity
6.
Mol Pharmacol ; 66(4): 880-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15229298

ABSTRACT

The presence of highly conserved amino acid stretches in G protein-coupled receptors (GPCRs) usually predicts an important role in receptor function. Considerable attention has therefore been focused on the involvement of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif at the cytoplasmic end of transmembrane domain 3 in the regulation of GPCR conformational states and/or the mediation of G protein activation. In the present study, we investigated the role of Glu129 and Arg130 in the ERY of thromboxane A2 receptor alpha (TPalpha) in transfected human embryonic kidney 293 cells. We show that no conservative or nonconservative substitutions of Glu129 and Arg130 generated a constitutively active TPalpha mutant, but a nonconservative mutation of Arg130 (R130V) yielded a mutant receptor with significantly impaired 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619)-induced accumulation of inositol phosphates (IPs). This loss-of-function phenotype seems to be caused by the uncoupling of the TPalpha receptor from Gq, as demonstrated by the loss of high-affinity agonist binding, and not by receptor internalization, as shown by localization studies with the R130V-green fluorescent protein fusion protein. It is interesting to note that U46619-induced activation of the nonconservative E129V mutant stimulated the production of IPs with a approximately 10-fold lower EC50 and a approximately 2-fold higher Emax than in the wild-type receptor. Collectively, these data demonstrate that, unlike other GPCRs, mutations of Glu129 do not induce constitutive activity, whereas Arg130 is involved in G protein coupling or recognition, and they suggest the existence within class A GPCRs of at least two different subclasses that make different uses of the highly conserved E/DRY motif.


Subject(s)
Conserved Sequence/genetics , Receptors, G-Protein-Coupled/physiology , Receptors, Thromboxane A2, Prostaglandin H2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cells, Cultured , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Thromboxane A2, Prostaglandin H2/agonists , Receptors, Thromboxane A2, Prostaglandin H2/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , Transfection
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