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Myocilin Interacts with Syntrophins and Is Member of Dystrophin-associated Protein Complex*

  • Myung Kuk Joe
    Affiliations
    Retinal Ganglion Cell Biology Section, Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892
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  • Changwon Kee
    Affiliations
    Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
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  • Stanislav I. Tomarev
    Correspondence
    To whom correspondence should be addressed: Retinal Ganglion Cell Biology Section, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, NIH, Bldg. 6, Rm. 212A, 6 Center Dr., Bethesda, MD 20892. Tel.: 301-496-8524;
    Affiliations
    Retinal Ganglion Cell Biology Section, Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892
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  • Author Footnotes
    * This work was supported, in whole or in part, by the National Institutes of Health, NEI, Intramural Research Program.
    This article contains supplemental Figs. 1–3.
Open AccessPublished:February 25, 2012DOI:https://doi.org/10.1074/jbc.M111.224063
      Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.

      Introduction

      Myocilin was identified in a human trabecular meshwork cell line as a glucocorticoid-induced protein more than 10 years ago (
      • Polansky J.R.
      • Fauss D.J.
      • Chen P.
      • Chen H.
      • Lütjen-Drecoll E.
      • Johnson D.
      • Kurtz R.M.
      • Ma Z.D.
      • Bloom E.
      • Nguyen T.D.
      Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product.
      ). Subsequent experiments demonstrated that myocilin belongs to a family of olfactomedin domain-containing proteins that consists of 13 members in mammals (
      • Tomarev S.I.
      • Nakaya N.
      Olfactomedin domain-containing proteins. Possible mechanisms of action and functions in normal development and pathology.
      ). Most of the olfactomedin domain-containing proteins, including myocilin, are secreted glycoproteins that demonstrate specific expression patterns. The N-terminal region of myocilin contains a leucine zipper, which is part of two coiled-coil domains, whereas the C-terminal region contains the olfactomedin domain. Myocilin is able to form dimers and multimers, and the N-terminal region of myocilin is critical for dimerization (
      • Nguyen T.D.
      • Chen P.
      • Huang W.D.
      • Chen H.
      • Johnson D.
      • Polansky J.R.
      Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells.
      ,
      • Fautsch M.P.
      • Johnson D.H.
      Characterization of myocilin-myocilin interactions.
      ,
      • Torrado M.
      • Trivedi R.
      • Zinovieva R.
      • Karavanova I.
      • Tomarev S.I.
      Optimedin. A novel olfactomedin-related protein that interacts with myocilin.
      ,
      • Wentz-Hunter K.
      • Ueda J.
      • Yue B.Y.
      Protein interactions with myocilin.
      ).
      It is now well established that mutations in the myocilin (MYOC) gene may lead to glaucoma and are found in more than 10% of juvenile open angle glaucoma cases and in 3–4% of patients with adult onset primary open angle glaucoma (
      • Fingert J.H.
      • Ying L.
      • Swiderski R.E.
      • Nystuen A.M.
      • Arbour N.C.
      • Alward W.L.
      • Sheffield V.C.
      • Stone E.M.
      Characterization and comparison of the human and mouse GLC1A glaucoma genes.
      ,
      • Stone E.M.
      • Fingert J.H.
      • Alward W.L.
      • Nguyen T.D.
      • Polansky J.R.
      • Sunden S.L.
      • Nishimura D.
      • Clark A.F.
      • Nystuen A.
      • Nichols B.E.
      • Mackey D.A.
      • Ritch R.
      • Kalenak J.W.
      • Craven E.R.
      • Sheffield V.C.
      Identification of a gene that causes primary open angle glaucoma.
      ,
      • Kwon Y.H.
      • Fingert J.H.
      • Kuehn M.H.
      • Alward W.L.
      Primary open-angle glaucoma.
      ,
      • Adam M.F.
      • Belmouden A.
      • Binisti P.
      • Brézin A.P.
      • Valtot F.
      • Béchetoille A.
      • Dascotte J.C.
      • Copin B.
      • Gomez L.
      • Chaventré A.
      • Bach J.F.
      • Garchon H.J.
      Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin homology domain of TIGR in familial open-angle glaucoma.
      ). Glaucoma is one of the leading causes of irreversible blindness in the world. It is estimated to affect more than 60 million and blind about 4.5 million people worldwide (
      • Quigley H.A.
      • Broman A.T.
      The number of people with glaucoma worldwide in 2010 and 2020.
      ). Primary open angle glaucoma is the most common form of glaucoma. More than 70 glaucoma-causing mutations in the MYOC gene have been identified, and greater than 90% of them are located in the region encoding the olfactomedin domain. Mutations causing a severe glaucoma phenotype lead to the retention of myocilin in the endoplasmic reticulum and prevent its secretion. Moreover, secretion of wild-type myocilin is impeded in the presence of mutated myocilin protein (
      • Jacobson N.
      • Andrews M.
      • Shepard A.R.
      • Nishimura D.
      • Searby C.
      • Fingert J.H.
      • Hageman G.
      • Mullins R.
      • Davidson B.L.
      • Kwon Y.H.
      • Alward W.L.
      • Stone E.M.
      • Clark A.F.
      • Sheffield V.C.
      Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.
      ,
      • Gobeil S.
      • Rodrigue M.A.
      • Moisan S.
      • Nguyen T.D.
      • Polansky J.R.
      • Morissette J.
      • Raymond V.
      Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants.
      ,
      • Malyukova I.
      • Lee H.S.
      • Fariss R.N.
      • Tomarev S.I.
      Mutated mouse and human myocilins have similar properties and do not block general secretory pathway.
      ,
      • Sohn S.
      • Hur W.
      • Joe M.K.
      • Kim J.H.
      • Lee Z.W.
      • Ha K.S.
      • Kee C.
      Expression of wild-type and truncated myocilins in trabecular meshwork cells. Their subcellular localizations and cytotoxicities.
      ). Accumulation of mutated myocilin in the endoplasmic reticulum may be deleterious for cells, making them more sensitive to oxidative stress leading to cell death (
      • Joe M.K.
      • Sohn S.
      • Hur W.
      • Moon Y.
      • Choi Y.R.
      • Kee C.
      Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.
      ,
      • Liu Y.
      • Vollrath D.
      Reversal of mutant myocilin non-secretion and cell killing. Implications for glaucoma.
      ,
      • Joe M.K.
      • Tomarev S.I.
      Expression of myocilin mutants sensitizes cells to oxidative stress-induced apoptosis. Implication for glaucoma pathogenesis.
      ).
      The MYOC gene is highly expressed in the trabecular meshwork, iris, ciliary body, sclera, and retinal pigmented epithelial cells, with lower levels of expression observed in skeletal muscle, mammary gland, thymus, and testis (
      • Torrado M.
      • Trivedi R.
      • Zinovieva R.
      • Karavanova I.
      • Tomarev S.I.
      Optimedin. A novel olfactomedin-related protein that interacts with myocilin.
      ,
      • Stone E.M.
      • Fingert J.H.
      • Alward W.L.
      • Nguyen T.D.
      • Polansky J.R.
      • Sunden S.L.
      • Nishimura D.
      • Clark A.F.
      • Nystuen A.
      • Nichols B.E.
      • Mackey D.A.
      • Ritch R.
      • Kalenak J.W.
      • Craven E.R.
      • Sheffield V.C.
      Identification of a gene that causes primary open angle glaucoma.
      ,
      • Adam M.F.
      • Belmouden A.
      • Binisti P.
      • Brézin A.P.
      • Valtot F.
      • Béchetoille A.
      • Dascotte J.C.
      • Copin B.
      • Gomez L.
      • Chaventré A.
      • Bach J.F.
      • Garchon H.J.
      Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin homology domain of TIGR in familial open-angle glaucoma.
      ,
      • Tomarev S.I.
      • Tamm E.R.
      • Chang B.
      Characterization of the mouse Myoc/Tigr gene.
      ). Available data suggest that the presence of wild-type myocilin is not critical for normal development and survival. Mice heterozygous and homozygous for a targeted null mutation in myocilin do not have a detectable phenotype (
      • Kim B.S.
      • Savinova O.V.
      • Reedy M.V.
      • Martin J.
      • Lun Y.
      • Gan L.
      • Smith R.S.
      • Tomarev S.I.
      • John S.W.
      • Johnson R.L.
      Targeted disruption of the myocilin gene (Myoc) suggests that human glaucoma-causing mutations are gain of function.
      ). Similarly, it has been reported that an elderly woman homozygous for the Arg46Stop mutation in the MYOC gene do not develop identifiable pathologies (
      • Lam D.S.
      • Leung Y.F.
      • Chua J.K.
      • Baum L.
      • Fan D.S.
      • Choy K.W.
      • Pang C.P.
      Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma.
      ).
      The functions of wild-type myocilin are still not very clear. One approach to the elucidation of protein functions includes the identification of proteins interacting with the protein of interest or the identification of protein complexes containing the protein of interest. Recently, we demonstrated that myocilin, similar to Wnt proteins, interacts with cysteine-rich domains of several frizzled receptors and secreted frizzled-related proteins as well as with Wnt inhibitory factor WIF-1 (
      • Kwon H.S.
      • Lee H.S.
      • Ji Y.
      • Rubin J.S.
      • Tomarev S.I.
      Myocilin is a modulator of Wnt signaling.
      ). Myocilin may modify the organization of the actin cytoskeleton, stimulating the formation of stress fibers, and this may be essential for the contractility of the trabecular meshwork and the regulation of intraocular pressure (
      • Kwon H.S.
      • Lee H.S.
      • Ji Y.
      • Rubin J.S.
      • Tomarev S.I.
      Myocilin is a modulator of Wnt signaling.
      ). We suggested that myocilin may serve as a modulator of Wnt signaling and that other family members or Wnt proteins may compensate for an absence of myocilin by performing its functions (
      • Kwon H.S.
      • Lee H.S.
      • Ji Y.
      • Rubin J.S.
      • Tomarev S.I.
      Myocilin is a modulator of Wnt signaling.
      ).
      In the present work, we demonstrate that myocilin is part of the dystrophin-associated protein complex (DAPC)
      The abbreviations used are: DAPC
      dystrophin-associated protein complex
      α1-Syn
      α1-syntrophin
      DG
      dystroglycan
      nNOS
      neuronal nitric-oxide synthase
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      TA
      tibialis anterior
      WGA
      wheat germ agglutinin
      mTOR
      mammalian target of rapamycin.
      in mouse skeletal muscle and in differentiating C2C12 cells forming myotubes. Myocilin interacts with α1-syntrophin (α1-Syn), a cytoplasmic component of the DAPC, that serves as a scaffolding adapter. Overexpression of myocilin in transgenic mice leads to a redistribution of some DAPC proteins and increased phosphorylation of Akt, mTOR, and Forkhead box O-class 3 (FoxO3) transcription factor, the key regulators of muscle size. Moreover, the muscle size was increased in transgenic mice compared with wild-type mice. We suggest that myocilin is one of the regulators of muscle hypertrophy pathways.

      DISCUSSION

      Glaucoma-causing mutations in the MYOC gene were first identified more than 10 years ago (
      • Stone E.M.
      • Fingert J.H.
      • Alward W.L.
      • Nguyen T.D.
      • Polansky J.R.
      • Sunden S.L.
      • Nishimura D.
      • Clark A.F.
      • Nystuen A.
      • Nichols B.E.
      • Mackey D.A.
      • Ritch R.
      • Kalenak J.W.
      • Craven E.R.
      • Sheffield V.C.
      Identification of a gene that causes primary open angle glaucoma.
      ,
      • Adam M.F.
      • Belmouden A.
      • Binisti P.
      • Brézin A.P.
      • Valtot F.
      • Béchetoille A.
      • Dascotte J.C.
      • Copin B.
      • Gomez L.
      • Chaventré A.
      • Bach J.F.
      • Garchon H.J.
      Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin homology domain of TIGR in familial open-angle glaucoma.
      ). Since then, the potential for myocilin to be used as a molecular tool to study glaucoma has stimulated active investigation into the function(s) of myocilin protein. However, despite these efforts, the role of myocilin in ocular and non-ocular tissues remains elusive. There is a growing number of cases showing that mutations in interacting proteins may produce similar phenotypes and that protein-protein interactions may be used to identify new candidate genes for diseases (
      • Oti M.
      • Snel B.
      • Huynen M.A.
      • Brunner H.G.
      Predicting disease genes using protein-protein interactions.
      ). Therefore, multiple attempts have been made to identify proteins that interact with myocilin and several candidate proteins belonging to different functional classes (extracellular matrix, cytoskeleton, cell signaling and metabolism, and membrane proteins) have been identified (
      • Torrado M.
      • Trivedi R.
      • Zinovieva R.
      • Karavanova I.
      • Tomarev S.I.
      Optimedin. A novel olfactomedin-related protein that interacts with myocilin.
      ,
      • Wentz-Hunter K.
      • Ueda J.
      • Yue B.Y.
      Protein interactions with myocilin.
      ,
      • Joe M.K.
      • Sohn S.
      • Choi Y.R.
      • Park H.
      • Kee C.
      Identification of flotillin-1 as a protein interacting with myocilin. Implications for the pathogenesis of primary open-angle glaucoma.
      ,
      • Fautsch M.P.
      • Vrabel A.M.
      • Johnson D.H.
      The identification of myocilin-associated proteins in the human trabecular meshwork.
      ). In many cases, interaction of myocilin with other proteins could not be confirmed by independent techniques (
      • Fautsch M.P.
      • Vrabel A.M.
      • Johnson D.H.
      The identification of myocilin-associated proteins in the human trabecular meshwork.
      ), and continuing work should be undertaken to confirm the specificity of these interactions. Using independent techniques, including a yeast two-hybrid assay and co-immunoprecipitation from cultured cells and native tissues, we demonstrate here that myocilin interacts with α1-Syn. Our preliminary data indicate that in other tissues, myocilin may also interact with β-syntrophin.
      M. K. Joe and S. I. Tomarev, unpublished observations.
      At first glance, an interaction between secretory (myocilin) and cytoplasmic (syntrophin) proteins seems unusual. However, accumulating evidence demonstrates that many signal peptide-containing proteins may have distinct functions outside of the secretory pathway. For example, endoplasmic reticulum-resident protein calreticulin was identified as a binding protein to the cytoplasmic α-subunits of integrin receptors (
      • Leung-Hagesteijn C.Y.
      • Milankov K.
      • Michalak M.
      • Wilkins J.
      • Dedhar S.
      Cell attachment to extracellular matrix substrates is inhibited upon down-regulation of expression of calreticulin, an intracellular integrin α-subunit-binding protein.
      ), the DNA-binding domain of a nuclear receptor (
      • Burns K.
      • Duggan B.
      • Atkinson E.A.
      • Famulski K.S.
      • Nemer M.
      • Bleackley R.C.
      • Michalak M.
      Modulation of gene expression by calreticulin binding to the glucocorticoid receptor.
      ), and p21 mRNA (
      • Iakova P.
      • Wang G.L.
      • Timchenko L.
      • Michalak M.
      • Pereira-Smith O.M.
      • Smith J.R.
      • Timchenko N.A.
      Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate.
      ) through independent studies. Alternative cellular localizations would provide functional diversity from a single protein through the interaction with different molecules that are present at the different cellular compartments. Data presented here suggest that myocilin is not secreted from muscle cells. The absence of myocilin secretion is not a unique property of muscle cells; myocilin is also not secreted from trabecular meshwork cells growing in culture in the presence of different concentrations of serum (
      • Resch Z.T.
      • Hann C.R.
      • Cook K.A.
      • Fautsch M.P.
      Aqueous humor rapidly stimulates myocilin secretion from human trabecular meshwork cells.
      ) and the human breast adenocarcinoma MCF7 cell line (
      • Hardy K.M.
      • Hoffman E.A.
      • Gonzalez P.
      • McKay B.S.
      • Stamer W.D.
      Extracellular trafficking of myocilin in human trabecular meshwork cells.
      ). NIH3T3 cells transfected with myocilin constructs do not secret myocilin, whereas transfected HEK293 and COS7 cells efficiently secret myocilin in the same conditions (
      • Malyukova I.
      • Lee H.S.
      • Fariss R.N.
      • Tomarev S.I.
      Mutated mouse and human myocilins have similar properties and do not block general secretory pathway.
      ). Published results indicate that suppression of myocilin secretion does not dramatically affect general secretion in vivo and in vitro (
      • Malyukova I.
      • Lee H.S.
      • Fariss R.N.
      • Tomarev S.I.
      Mutated mouse and human myocilins have similar properties and do not block general secretory pathway.
      ,
      • Hardy K.M.
      • Hoffman E.A.
      • Gonzalez P.
      • McKay B.S.
      • Stamer W.D.
      Extracellular trafficking of myocilin in human trabecular meshwork cells.
      ,
      • Zhou Y.
      • Grinchuk O.
      • Tomarev S.I.
      Transgenic mice expressing the Tyr437His mutant of human myocilin protein develop glaucoma.
      ). Myocilin may have a distinct function in the cytoplasm that is different from its functions in the extracellular space.
      Syntrophins share a similar domain organization and functional conservation among the isotypes. The syntrophins interact with various components of the DAPC and bind to the C-terminal region of dystrophin (
      • Ehmsen J.
      • Poon E.
      • Davies K.
      The dystrophin-associated protein complex.
      ,
      • Piluso G.
      • Mirabella M.
      • Ricci E.
      • Belsito A.
      • Abbondanza C.
      • Servidei S.
      • Puca A.A.
      • Tonali P.
      • Puca G.A.
      • Nigro V.
      γ1- and γ2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells.
      ,
      • Ahn A.H.
      • Freener C.A.
      • Gussoni E.
      • Yoshida M.
      • Ozawa E.
      • Kunkel L.M.
      The three human syntrophin genes are expressed in diverse tissues, have distinct chromosomal locations, and each bind to dystrophin and its relatives.
      ). α1-Syn is a cytoplasmic component of the DAPC that serves as a scaffolding adapter. The PDZ domain of α1-Syn binds to several membrane proteins, including nNOS, sodium channels, kinases, and aquaporin 4 (
      • Waite A.
      • Tinsley C.L.
      • Locke M.
      • Blake D.J.
      The neurobiology of the dystrophin-associated glycoprotein complex.
      ,
      • Ehmsen J.
      • Poon E.
      • Davies K.
      The dystrophin-associated protein complex.
      ). The PDZ domain of α1-Syn most probably is not essential for interactions with myocilin because this domain was absent in the yeast clone identified by an yeast two-hybrid screen and because myocilin and the C-terminal region of α1-Syn without the PDZ domain were co-localized well in mammalian cells transfected with corresponding constructs.3 The N-terminal domain of myocilin (without the olfactomedin domain) is essential for interaction with α1-Syn (Fig. 2).
      It has been shown that expression of α1-Syn and its binding to the components of DAPC may be modified in myofibers from patients with common forms of muscle dystrophies (
      • Wakayama Y.
      • Inoue M.
      • Kojima H.
      • Jimi T.
      • Yamashita S.
      • Kumagai T.
      • Shibuya S.
      • Hara H.
      • Oniki H.
      Altered α1-syntrophin expression in myofibers with Duchenne and Fukuyama muscular dystrophies.
      ,
      • Nakamori M.
      • Kimura T.
      • Kubota T.
      • Matsumura T.
      • Sumi H.
      • Fujimura H.
      • Takahashi M.P.
      • Sakoda S.
      Aberrantly spliced α-dystrobrevin alters α-syntrophin binding in myotonic dystrophy type 1.
      ). It has been suggested that changes in the recruitment of α1-Syn to the sarcolemma might lead to a disturbance of intracellular signaling and affect muscle function (
      • Nakamori M.
      • Kimura T.
      • Kubota T.
      • Matsumura T.
      • Sumi H.
      • Fujimura H.
      • Takahashi M.P.
      • Sakoda S.
      Aberrantly spliced α-dystrobrevin alters α-syntrophin binding in myotonic dystrophy type 1.
      ). Overexpression of myocilin in the muscles of transgenic mice also led to a redistribution of some DAPC-associated proteins, including syntrophin, and may have affected intracellular signaling. Oppositely, the absence of myocilin led to a moderate reduction in the amount of syntrophin associated with dystrophin and reduced levels of phospho-Akt in the muscle of myocilin null mice compared with wild-type littermates.
      We demonstrated that overexpression of myocilin in skeletal muscles led to muscle hypertrophy. Skeletal muscle hypertrophy results from an increase in the diameter of muscle fibers that are formed after the differentiation and fusion of myoblast precursors. Several signaling pathways have been implicated in muscle hypertrophy, including the insulin-like growth factor 1/phosphoinositide/Akt signaling pathway, which contributes to regulation of skeletal muscle fiber size (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      Mediation of IGF-1-induced skeletal myotube hypertrophy by PI3K/Akt/mTOR and PI3K/Akt/GSK3 pathways.
      ). Activation of this cascade keeps the FoxO3 transcription factor in an inactive (phosphorylated) state and sequesters FoxO3 in the cytosol (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy.
      ). Activation of FoxO3 stimulates transcription of a number of genes, including atrophy-linked ubiquitin ligases astrogin-1 and MuRF-1 (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy.
      ). Recent data demonstrate that transcription factor JunB is important for the maintenance of adult muscle size and can induce rapid hypertrophy and block atrophy (
      • Raffaello A.
      • Milan G.
      • Masiero E.
      • Carnio S.
      • Lee D.
      • Lanfranchi G.
      • Goldberg A.L.
      • Sandri M.
      JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy.
      ). However, although JunB blocks FoxO3 binding to atrogin-1 and MuRF-1 promoters, it induces hypertrophy independently of the Akt signaling pathway (
      • Raffaello A.
      • Milan G.
      • Masiero E.
      • Carnio S.
      • Lee D.
      • Lanfranchi G.
      • Goldberg A.L.
      • Sandri M.
      JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy.
      ). We found that myocilin-induced muscle hypertrophy involves activation of the Akt signaling pathway and in this respect is more similar to insulin-like growth factor-1-induced skeletal myotube hypertrophy (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      Mediation of IGF-1-induced skeletal myotube hypertrophy by PI3K/Akt/mTOR and PI3K/Akt/GSK3 pathways.
      ). Extracellular myocilin may serve as a modulator of Wnt signaling (
      • Kwon H.S.
      • Lee H.S.
      • Ji Y.
      • Rubin J.S.
      • Tomarev S.I.
      Myocilin is a modulator of Wnt signaling.
      ) and possess an ability to activate the integrin-FAK-PI3K-Akt signaling pathway (
      • Kwon H.S.
      • Tomarev S.I.
      Myocilin, a glaucoma-associated protein, promotes cell migration through activation of integrin-focal adhesion kinase-serine/threonine kinase signaling pathway.
      ). Because myocilin is not secreted from muscle cells, we believe that activation of the Akt signaling pathway in transgenic mice occurs mainly through the action of intracellular myocilin. One function of DAPC is to provide a connection between extracellular matrix and intracellular signaling. An elevated level of DAPC-associated myocilin leads to a redistribution of DAPC components that may stabilize DAPC and lead to enhanced binding of extracellular ligands, such as laminin, to DAPC (see Fig. 5). This in turn leads to the activation of Akt and phosphorylation of FoxO3a. Extracellular myocilin probably does not play a prominent role in skeletal muscles, although we cannot completely exclude participation of extracellular myocilin in the observed effects.
      The mean cross-section area of muscle fibers was increased by 36% in myocilin transgenic mice compared with their wild-type littermates (Fig. 7D). This number is similar to that observed for mouse muscle fibers overexpressing JunB for 9 days (about 40% increase) (
      • Raffaello A.
      • Milan G.
      • Masiero E.
      • Carnio S.
      • Lee D.
      • Lanfranchi G.
      • Goldberg A.L.
      • Sandri M.
      JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy.
      ). It has been suggested that the JunB increase can cause marked hypertrophy and can block the rapid loss of muscle mass in bed-ridden patients (
      • Raffaello A.
      • Milan G.
      • Masiero E.
      • Carnio S.
      • Lee D.
      • Lanfranchi G.
      • Goldberg A.L.
      • Sandri M.
      JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy.
      ). Although more research is necessary, we speculate that increasing myocilin levels in skeletal muscle may also be considered as a potential new therapeutic approach to combat the muscle loss during certain diseases or in the elderly. Although myocilin was discovered as a gene that is up-regulated in the trabecular meshwork as a result of glucocorticoid treatment, myocilin was not identified among genes induced in skeletal muscles by the dexamethasone treatment (
      • Wu Y.
      • Zhao W.
      • Zhao J.
      • Zhang Y.
      • Qin W.
      • Pan J.
      • Bauman W.A.
      • Blitzer R.D.
      • Cardozo C.
      REDD1 is a major target of testosterone action in preventing dexamethasone-induced muscle loss.
      ,
      • Carraro L.
      • Ferraresso S.
      • Cardazzo B.
      • Romualdi C.
      • Montesissa C.
      • Gottardo F.
      • Patarnello T.
      • Castagnaro M.
      • Bargelloni L.
      Expression profiling of skeletal muscle in young bulls treated with steroidal growth promoters.
      ,
      • Komamura K.
      • Shirotani-Ikejima H.
      • Tatsumi R.
      • Tsujita-Kuroda Y.
      • Kitakaze M.
      • Miyatake K.
      • Sunagawa K.
      • Miyata T.
      Differential gene expression in the rat skeletal and heart muscle in glucocorticoid-induced myopathy. Analysis by microarray.
      ). In skeletal muscles, dexamethasone treatment changed the expression pattern of hundreds of genes and could cause muscle atrophy (see Refs.
      • Schakman O.
      • Gilson H.
      • Thissen J.P.
      Mechanisms of glucocorticoid-induced myopathy.
      and
      • Hasselgren P.O.
      • Alamdari N.
      • Aversa Z.
      • Gonnella P.
      • Smith I.J.
      • Tizio S.
      Corticosteroids and muscle wasting. Role of transcription factors, nuclear cofactors, and hyperacetylation.
      for recent reviews). In transgenic mice, myocilin overexpression is due to the increased copy number of the Myoc gene. Up-regulation of myocilin in the absence of glucocorticoid increase leads to changes that are opposite to those observed after glucocorticoid treatment at both molecular (down-regulation of MuRF1 and astrogin-1 versus their up-regulation) and tissue levels (muscle hypertrophy versus atrophy).
      Experiments reported in this paper were performed using skeletal muscles because of the relative abundance of biological material for biochemical testing. Myocilin is highly expressed in the tissues of the eye angle, including the trabecular meshwork, sclera, iris, and ciliary muscle (
      • Torrado M.
      • Trivedi R.
      • Zinovieva R.
      • Karavanova I.
      • Tomarev S.I.
      Optimedin. A novel olfactomedin-related protein that interacts with myocilin.
      ,
      • Senatorov V.
      • Malyukova I.
      • Fariss R.
      • Wawrousek E.F.
      • Swaminathan S.
      • Sharan S.K.
      • Tomarev S.
      Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice.
      ). The state of ciliary muscle and its contractility are important for the architecture of the conventional trabecular meshwork outflow pathway and unconventional uveoscleral outflow pathway (
      • Tamm E.R.
      The trabecular meshwork outflow pathways. Structural and functional aspects.
      ,
      • Alm A.
      • Nilsson S.F.
      Uveoscleral outflow. A review.
      ). Modulations of these outflow pathways may lead to changes in intraocular pressure.
      In summary, myocilin interacts with α1-Syn and co-immunoprecipitates with several DAPC components, including dystrophin and dystroglycan. Overexpression of myocilin in transgenic mice leads to redistribution of several DAPC components and increased phosphorylation of Akt, a key regulator of muscle size. Accordingly, muscle size was increased in transgenic mice compared with wild-type mice. We suggest that myocilin is a regulator of muscle hypertrophy/atrophy pathways and that the stabilization of DAPC and increased interaction with laminin may contribute to this regulation. Future studies will be aimed at elucidating the roles of myocilin and DAPC in the eye drainage structures to determine how the elevated levels of myocilin may change the DAPC complex in these structures and whether overexpression of myocilin affects contractility of the ciliary muscle.

      Acknowledgments

      We thank Dr. Heung Sun Kwon for myocilin constructs and Dr. Thomas V. Johnson and Nicholas Dekorver for critical reading of the manuscript.

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