Genetic deletion of microRNA biogenesis in muscle cells reveals a hierarchical non-clustered network that controls focal adhesion signaling during muscle regeneration

ABSTRACTTensins are a family of focal adhesion proteins consisting of four members in mammals (TNS1, TNS2, TNS3 and TNS4). Their multiple domains and activities contribute to the molecular linkage between the extracellular matrix and cytoskeletal networks, as well as mediating signal transduction pathways, leading to a variety of physiological processes, including cell proliferation, attachment, migration and mechanical sensing in a cell. Tensins are required for maintaining normal tissue structures and functions, especially in the kidney and heart, as well as in muscle regeneration, in animals. This Review discusses our current understanding of the domain functions and biological roles of tensins in cells and mice, as well as highlighting their relevance to human diseases.

Download Full-text

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0821 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1409-1419 ◽

Cited By ~ 29

Author(s):

Luca Madaro ◽

Valeria Marrocco ◽

Piera Fiore ◽

Paola Aulino ◽

Piera Smeriglio ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Muscle Regeneration ◽

Myoblast Fusion ◽

Dominant Negative Mutant ◽

Postnatal Life ◽

Mutant Form ◽

In Vitro Differentiation ◽

Freeze Injury

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKCθ signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.

Download Full-text

Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00541.2012 ◽

2013 ◽

Vol 305 (2) ◽

pp. E183-E193 ◽

Cited By ~ 45

Author(s):

Hannah Crossland ◽

Abid A. Kazi ◽

Charles H. Lang ◽

James A. Timmons ◽

Philippe Pierre ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Growth ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Muscle Hypertrophy ◽

Kinase Activity ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Cell Phenotype ◽

Igf I

Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C2C12 skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr397 phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.

Download Full-text

Influences of surface chemistry and swelling of salt-treated polyelectrolyte multilayers on migration of smooth muscle cells

Journal of The Royal Society Interface ◽

10.1098/rsif.2012.0546 ◽

2012 ◽

Vol 9 (77) ◽

pp. 3455-3468 ◽

Cited By ~ 24

Author(s):

Lulu Han ◽

Zhengwei Mao ◽

Jindan Wu ◽

Yuying Zhang ◽

Changyou Gao

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Surface Chemistry ◽

Smooth Muscle Cells ◽

Focal Adhesion ◽

Chemical Properties ◽

Muscle Cells ◽

Cellular Migration ◽

Gene Expressions ◽

And Migration

The cell migration plays a crucial role in a variety of physiological and pathological processes and can be regulated by the cell–substrate interactions. We found previously that the poly(sodium 4-styrenesulphonate) (PSS)/poly(diallyldimethylammonium) chloride (PDADMAC) multilayers post-treated in 1–5 M NaCl solutions result in continuous changes of their physico-chemical properties such as thickness, chemical composition, surface charge, swelling ratio and wettability. In this study, the responses of human smooth muscle cells (SMCs) on these salt-treated multilayers, particularly the governing factors of cellular migration that offer principles for designing therapeutics and implants, were disclosed. The cell migration rate was slowest on the 3 M NaCl-treated multilayers, which was comparable with that on tissue culture plates, but it was highest on 5 M NaCl-treated multilayers. To elucidate the intrinsic mechanisms, cell adhesion, proliferation, adhesion and related gene expressions were further investigated. The SMCs preferred to attach, spread and proliferate on the PSS-dominated surfaces with well-organized focal adhesion and actin fibres, especially on the 3 M NaCl-treated multilayers, while were kept round and showed low viability on the PDADMAC-dominated surfaces. The relative mRNA expression levels of adhesion-related genes such as fibronectin, laminin and focal adhesion kinase, and migration-related genes such as myosin IIA and Cdc42 were compared to explain the different cellular behaviours. These results reveal that the surface chemistry and the swelling of the salt-treated multilayers govern the cell migration behaviours.

Download Full-text

Angiotensin II Stimulates Tyrosine Phosphorylation of the Focal Adhesion-associated Protein Paxillin in Aortic Smooth Muscle Cells

Journal of Biological Chemistry ◽

10.1074/jbc.270.9.4401 ◽

1995 ◽

Vol 270 (9) ◽

pp. 4401-4404 ◽

Cited By ~ 70

Author(s):

Isabelle Leduc ◽

Sylvain Meloche

Keyword(s):

Smooth Muscle ◽

Angiotensin Ii ◽

Smooth Muscle Cells ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Muscle Cells ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle

Download Full-text

R-spondin1 regulates muscle progenitor cell fusion through control of antagonist Wnt signaling pathways

10.1101/063669 ◽

2016 ◽

Author(s):

Floriane Lacour ◽

Elsa Vezin ◽

Florian Bentzinger ◽

Marie-Claude Sincennes ◽

Robert D. Mitchell ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Stem Cell ◽

Wnt Signaling ◽

Signaling Pathways ◽

Muscle Regeneration ◽

Muscle Cells ◽

Skeletal Muscle Regeneration ◽

Acute Injury ◽

Canonical Wnt

SUMMARYTissue regeneration requires the selective activation and repression of specific signaling pathways in stem cells. As such, the Wnt signaling pathways have been shown to control stem cell fate. In many cell types, the R-Spondin (Rspo) family of secreted proteins acts as potent activators of the canonical Wnt/β-catenin pathway. Here, we identify Rspo1 as a mediator of skeletal muscle tissue repair. Firstly we show that Rspo1-null muscles do not display any abnormalities at the basal level. However deletion of Rspo1 results in global alteration of muscle regeneration kinetics following acute injury. We found that muscle stem cells lacking Rspo1 show delayed differentiation. Transcriptome analysis further demonstrated that Rspo1 is required for the activation of Wnt/β-catenin target genes in muscle cells. Furthermore, muscle cells lacking Rspo1 fuse with a higher frequency than normal cells, leading to larger myotubes containing more nuclei both in vitro and in vivo. We found the increase in muscle fusion was dependent on up-regulation of non-canonical Wnt7a/Fzd7/Rac1 signaling. We conclude that antagonistic control of canonical and non-canonical Wnt signaling pathways by Rspo1 in muscle stem cell progeny is important for restitution of normal muscle architecture during skeletal muscle regeneration.

Download Full-text

αvβ3-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00475.2002 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1330-C1338 ◽

Cited By ~ 11

Author(s):

M. Sajid ◽

R. Zhao ◽

A. Pathak ◽

S. S. Smyth ◽

G. A. Stouffer

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Focal Adhesion ◽

Immunofluorescence Microscopy ◽

Adhesion Formation ◽

Focal Adhesions ◽

Muscle Cells ◽

Neointimal Formation ◽

Focal Adhesion Formation ◽

Integrin Antagonists

αvβ3-Integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because α-thrombin contributes to neointimal formation, we examined the hypothesis that αvβ3-integrins influence α-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed αvβ3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to α-thrombin were partially inhibited by anti-β3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that α-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by αvβ3 antagonists. β3-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. α-Thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). αvβ3-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from β3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, αvβ3-integrins play an important role in α-thrombin-induced proliferation and focal adhesion formation in RASMC.

Download Full-text

NO decreases phosphorylation of focal adhesion proteins via reduction of Ca in rat aortic smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.274.5.h1613 ◽

1998 ◽

Vol 274 (5) ◽

pp. H1613-H1619 ◽

Cited By ~ 4

Author(s):

Kuljeet Kaur ◽

Jian Yao ◽

Xiaolei Pan ◽

Carolyn Matthews ◽

Aviv Hassid

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Focal Adhesion ◽

Muscle Cells ◽

Ca Channel ◽

Tetraacetic Acid ◽

Adhesion Proteins ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle ◽

Focal Adhesion Proteins

Our laboratory has previously reported that the antimitogenic effect of nitric oxide (NO) in primary cultures of rat aortic smooth muscle cells may be attributed to activation of protein tyrosine phosphatase and dephosphorylation of protein phosphotyrosine [G. S. Dhaunsi, C. Matthews, K. Kaur, and A. Hassid. Am. J. Physiol. 272 ( Heart Circ. Physiol. 41): H1342–H1349, 1997]. The goal of the current study was to investigate the role of cytoplasmic Ca in this process and to identify protein substrates that are dephosphorylated by treatment with NO. Treatment of primary rat aortic smooth muscle cell cultures with the NO donor S-nitroso- N-acetylpenicillamine (SNAP) decreased cytoplasmic Ca levels and elicited phosphotyrosine dephosphorylation. Both effects were mimicked by the extracellular and intracellular Ca chelators ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA), respectively, and by the Ca channel blocker nifedipine. Conversely, elevation of cytoplasmic Ca via the use of the Ca ionophore A-23187 or high extracellular K+prevented or attenuated SNAP-induced dephosphorylation. Both BAPTA and nifedipine also decreased DNA synthesis, providing further evidence to link dephosphorylation to antimitogenesis. Two of the proteins dephosphorylated by treatment of cells with NO or EGTA were identified as the focal adhesion proteins, cortactin and paxillin. These results indicate that NO-induced dephosphorylation of protein phosphotyrosine is mediated by reduction of cytoplasmic Ca and suggest that dephosphorylation of focal adhesion proteins may be of relevance to the antimitogenic effect of NO.

Download Full-text