Proteasome Inhibitor (MG-132) Treatment of mdx Mice Rescues the Expression and Membrane Localization of Dystrophin and Dystrophin-Associated Proteins
2003; Elsevier BV; Volume: 163; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63523-7
ISSN1525-2191
AutoresGloria Bonuccelli, Federica Sotgia, William Schubert, David Park, Philippe G. Frank, Scott E. Woodman, Luigi Insabato, Michael Cammer, Carlo Minetti, Michael P. Lisanti,
Tópico(s)Autophagy in Disease and Therapy
ResumoDystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, β-dystroglycan, α-dystroglycan, and α-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD. Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, β-dystroglycan, α-dystroglycan, and α-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD. Duchenne muscular dystrophy (DMD) is one of the most prevalent and severe inherited diseases of childhood, characterized by progressive muscular wasting and weakness. The deficient gene product, dystrophin,1Hoffman EP Brown RH Kunkel LM Dystrophin: the protein product of the Duchenne muscular dystrophy locus.Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3737) Google Scholar is a peripheral membrane protein of ∼426 kd, which is expressed in muscle tissues and the brain. At the plasma membrane, dystrophin associates with a large multimeric complex, termed the dystrophin-glycoprotein complex (DGC).2Ervasti JM Campbell KP Membrane organization of the dystrophin-glycoprotein complex.Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1123) Google Scholar The DGC is composed of two subcomplexes: the dystroglycan complex (α and β subunits) and the sarcoglycan complex (α, β, γ, and δ subunits). The N-terminal region of dystrophin interacts directly with the cytoskeletal protein actin, while the dystrophin C-terminal domain binds to the plasma membrane through interactions with β-dystroglycan. As such, dystrophin is thought to provide a mechanical linkage between the intracellular cytoskeleton and the extracellular matrix. The dystrophin complex also interacts with neuronal-type nitric oxide synthase (nNOS), whose biological product, NO, regulates contraction in skeletal muscle.3Brenman JE Chao DS Xia H Aldape K Bredt DS Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (855) Google Scholar, 4Crosbie RH Yamada H Venzke DP Lisanti MP Campbell KP Caveolin-3 is not an essential component of the dystrophin glycoprotein complex.FEBS Lett. 1998; 427: 279-282Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Another protein associated with the DGC, although not essential for the biogenesis of the complex itself, is caveolin-3 (Cav-3), a member of the caveolin protein family.5Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells: caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (617) Google Scholar Caveolins are the main structural components of caveolae, which are cholesterol- and sphingolipid-rich vesicular invaginations of the plasma membrane.6Rothberg KG Heuser JE Donzell WC Ying Y Glenney JR Anderson RGW Caveolin, a protein component of caveolae membrane coats.Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1881) Google Scholar, 7Smart EJ Graf GA McNiven MA Sessa WC Engelman JA Scherer PE Okamoto T Lisanti MP Caveolins, liquid-ordered domains, and signal transduction.Mol Cell Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (926) Google Scholar Research on DMD has greatly benefited from the availability of a naturally occurring mouse model, known as mdx, in which a non-sense mutation (premature stop codon) in the dystrophin gene ablates the expression of the dystrophin protein product.8Bulfield G Siller WG Wight PAL Moore KJ X-chromosome linked muscular dystrophy (mdx) in the mouse.Proc Natl Acad Sci USA. 1984; 81: 1189-1192Crossref PubMed Scopus (1412) Google Scholar, 9Sicinski P Geng Y Ryder-Cook AS Barnard EA Darlison MG Barnard PJ The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.Science. 1989; 244: 1578-1580Crossref PubMed Scopus (1017) Google Scholar The mdx mouse is viable and fertile, and exhibits histological lesions typical of muscular dystrophy. Although the mdx mouse is a valuable model for DMD, muscular wastage progresses in a much milder fashion than as compared with humans. This difference could be due to compensatory mechanisms, such as increased muscle regeneration, or the functional replacement of dystrophin by utrophin. Utrophin, the ubiquitous homologue of dystrophin, is normally expressed at the sarcolemma of skeletal muscle fibers during fetal development, but is restricted to the neuromuscular and myotendinous junctions in adult skeletal muscle.10Deconinck AE Rafael JA Skinner JA Brown SC Potter AC Metzinger L Watt DJ Dickson JG Tinsley JM Davies KE Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.Cell. 1997; 90: 717-727Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar The complete loss of dystrophin perturbs the structural composition of the DGC, such that all members of the DGC complex are greatly reduced in skeletal muscle fibers from DMD patients and from mdx mice.11Campbell KP Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage.Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (763) Google Scholar The only exception is Cav-3, which was shown to be up-regulated by ∼2-fold in dystrophin-deficient skeletal muscle.12Vaghy PL Fang J Wu W Vaghy LP Increased caveolin-3 levels in mdx mouse muscles.FEBS Lett. 1998; 431: 125-127Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 13Repetto S Bado M Broda P Lucania G Masetti E Sotgia F Carbone I Pavan A Bonilla E Cordone G Lisanti MP Minetti C Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy.Biochem Biophys Res Commun. 1999; : 547-550Crossref PubMed Scopus (97) Google Scholar A lack of dystrophin is thought to cause sarcolemmal instability, which may render the dystrophin-glycoprotein complex more susceptible to proteolytic degradation.14Kamper A Rodemann HP Alterations of protein degradation and 2-D protein pattern in muscle cells of mdx and DMD origin.Biochem Biophys Res Commun. 1992; 189: 1484-1490Crossref PubMed Scopus (15) Google Scholar Similarly to other tissues, skeletal muscle has at least three different pathways for protein degradation: 1) proteolysis by lysosomal proteases, such as the cathepsins, 2) proteolysis by non-lysosomal Ca2+-dependent proteases, such as calpain, and 3) proteolysis by non-lysosomal ATP-ubiquitin-dependent proteases, eg, the multicatalytic protease complex (or proteasome). The ubiquitin-proteasome pathway is the major proteolytic system present in all eukaryotic cells, and degrades the substrates marked by attachment of many molecules of ubiquitin, a small 8-kd protein. The resulting ubiquitinated proteins are then recognized and degraded by a 2.4-MDa proteolytic complex, the 26S proteasome. The proteasome consists of a cylindrical 20S catalytic core particle, capped by two 19S regulatory complexes that control the access of substrates to the proteolytic chamber.15Kisselev A Goldberg A Proteasome inhibitors: from research tools to drug candidates.Chem Biol. 2001; 8: 739-758Abstract Full Text Full Text PDF PubMed Scopus (1011) Google Scholar Several lines of evidence have suggested that enhanced activation of proteolytic degradation pathways underlies the pathogenesis of various diseases, including skeletal muscle atrophy and muscular dystrophy.16Matthews W Driscoll J Tanaka K Ichihara A Goldberg AL Involvement of the proteasome in various degradative processes in mammalian cells.Proc Natl Acad Sci USA. 1989; 86: 2597-2601Crossref PubMed Scopus (142) Google Scholar, 17Solomon V Goldberg A Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts.J Biol Chem. 1996; 271: 26690-26697Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 18Tawa Jr, NE Odessey R Goldberg AL Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.J Clin Invest. 1997; 100: 197-203Crossref PubMed Scopus (265) Google Scholar, 19Gao Y Lecker S Post M Hietaranta A Li J Volk R Li M Sato K Saluja A Steer M Goldberg A Simons M Inhibition of ubiquitin-proteasome pathway-mediated I κB α degradation by a naturally occurring antibacterial peptide.J Clin Invest. 2000; 106: 439-448Crossref PubMed Scopus (162) Google Scholar Combaret and colleagues20Combaret L Taillandier D Voisin L Samuels SE Boespflug-Tanguy O Attaix D No alteration in gene expression of components of the ubiquitin-proteasome proteolytic pathway in dystrophin-deficient muscles.FEBS Lett. 1996; 393: 292-296Abstract Full Text PDF PubMed Scopus (41) Google Scholar have demonstrated that increased protein degradation in skeletal muscle from mdx mice and DMD patients correlates with elevated expression of the non-lysosomal protease calpain, but not with elevated mRNA levels of components of the proteasomal pathway. Conversely, Kumamoto and colleagues21Kumamoto T Fujimoto S Ito T Horinouchi H Ueyama H Tsuda T Proteasome expression in the skeletal muscles of patients with muscular dystrophy.Acta Neuropathol (Berl). 2000; 100: 595-602Crossref PubMed Scopus (55) Google Scholar have provided preliminary evidence that, in DMD patients, muscle fiber degradation is due to concomitant activation of the non-lysosomal calpain-mediated pathway and of the non-lysosomal ATP-ubiquitin dependent proteasome system, as assessed by immunohistochemical staining. As such, the role of the proteasomal pathway in dystrophin-deficient skeletal muscle degeneration still remains controversial. Over the last several years, an increasing body of evidence has emerged highlighting the function of the proteasomal machinery in maintaining normal muscle size and capacity, and has suggested that dysregulation of the proteasomal pathway might result in muscle pathology. The discovery of two muscle-specific ubiquitin ligases, which target proteins for degradation by the proteasomal pathway, has provided a greater understanding of the mechanisms underlying muscle atrophy. For example, adenovirus-mediated over-expression of these muscle-specific ubiquitin ligases produces muscle atrophy, whereas their genetic ablation resulted in resistance to muscle atrophy.22Bodine S Latres E Baumhueter S Lai V Nunez L Clarke B Poueymirou W Panaro F Na E Dharmarajan K Pan Z Valenzuela D DeChiara T Stitt T Yancopoulos G Glass D Identification of ubiquitin ligases required for skeletal muscle atrophy.Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2706) Google Scholar Previous studies from our laboratory23Galbiati F Volonté D Minetti C Bregman DB Lisanti MP Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation: treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3.J Biol Chem. 2000; 275: 37702-37711Crossref PubMed Scopus (88) Google Scholar have demonstrated that proteasomal degradation may be involved in the pathogenesis of a form of muscular dystrophy in humans, Limb-Girdle Muscular Dystrophy (LGMD-1C). LGMD-1C is an autosomal dominant form of muscular dystrophy caused by heterozygous mutations in the caveolin-3 gene.24Minetti C Sotgia F Bruno C Scartezzini P Broda P Bado M Masetti E Mazzocco P Egeo A Donati MA Volonté D Galbiati F Cordone G Bricarelli FD Lisanti MP Zara F Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.Nat Genet. 1998; 18: 365-368Crossref PubMed Scopus (499) Google Scholar In a heterologous cell system, LGMD-1C mutants of Cav-3 behave in a dominant-negative fashion, causing the retention of wild-type Cav-3 at the level of the Golgi complex.25Galbiati F Volonté D Minetti C Chu JB Lisanti MP Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C).J Biol Chem. 1999; 274: 25632-25641Crossref PubMed Scopus (139) Google Scholar Further analysis has demonstrated that LGMD-1C mutants of Cav-3 undergo ubiquitination and proteasomal degradation. Treatment of cultured cells with MG-132 could effectively block the dominant negative effect of these mutants and rescue the expression levels and the subcellular localization of wild-type Cav-3.23Galbiati F Volonté D Minetti C Bregman DB Lisanti MP Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation: treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3.J Biol Chem. 2000; 275: 37702-37711Crossref PubMed Scopus (88) Google Scholar The discovery of many synthetic and natural inhibitors of the proteasomal pathway has been extremely useful in the comprehension of the mechanisms underlying intracellular protein degradation.15Kisselev A Goldberg A Proteasome inhibitors: from research tools to drug candidates.Chem Biol. 2001; 8: 739-758Abstract Full Text Full Text PDF PubMed Scopus (1011) Google Scholar Most importantly, these new drugs may have very important in vivo applications, and may be instrumental in the pharmacological treatment of various diseases. Here we assess whether activation of the ubiquitin-proteasome proteolytic pathway underlies the rapid loss of muscle proteins in dystrophin-deficient skeletal muscle in vivo. Our findings may have implications for the pharmacological treatment of patients with DMD. Monoclonal antibodies directed against β-dystroglycan (NCL-b-DG), α-sarcoglycan (NCL-a-sarco) and dystrophin (NCL-DYS3) were purchased from Novocastra (Newcastle, United Kingdom). A monoclonal antibody for α-dystroglycan was from Upstate Biotechnology (Lake Placid, NY); a polyclonal antibody against neuronal nNOS was purchased from BD Transduction Laboratories; and anti-caveolin-3 IgG (mAb 265Song KS Scherer PE Tang Z-L Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells: caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (617) Google Scholar) was the gift of Dr. Roberto Campos-Gonzalez from BD Transduction Laboratories. The proteasome inhibitor MG-132 (CBZ-leucyl-leucyl-leucinal) was from Calbiochem (San Diego, CA); Evans blue dye (EBD) was from Sigma (St. Louis, MO); Alzet Minipumps were purchased from Alza Corp. (Palo Alto, CA). The calpain activity kit (using a fluorogenic substrate) was from Oncogene Research Products (San Diego, CA). Six-month-old male mdx (C57BL/10ScSn DMD mdx) mice, purchased from The Jackson Laboratory (JAX mice; Bar Harbor, ME), were used throughout this study. Skeletal muscle tissues were quickly dissected, flash-frozen in 2-methyl butane (isopentane) cooled in liquid nitrogen, and stored at −80°C until use. Localized administration was performed by injection of MG-132 into the gastrocnemius muscles of mdx mice. To visualize the injected muscle, MG-132 (final concentration of 20 μmol/L) was pre-mixed with 1% India ink in phosphate-buffered saline (PBS) for a total volume of 100 μl. Mice were sacrificed 24 hours after injection, and skeletal muscles were quickly isolated for further analysis. To systemically administer MG-132, we subcutaneously implanted Alzet Minipumps in the anterior back region of mdx mice. Experiments were conducted on 6-month-old mdx mice. For 8 days, we administrated either different concentrations of MG-132 (delivered at rate of either 1 μg, or 5 μg or 10 μg/kg/24 hours) or the inhibitor-diluent (PBS only), as a negative control. Skeletal muscle tissues were collected from untreated (PBS only) and MG-132-treated mdx mice for further analysis. Tissue samples were isolated from the gastrocnemius muscle, rapidly frozen in liquid nitrogen-cooled isopentane, and stored at −80°C. Unfixed sections (6-μm thick) of frozen skeletal muscle were blocked in PBS with 1% bovine serum albumin (BSA) for 1 hour at room temperature. Then, sections were incubated with the primary antibody for 1 hour at room temperature (diluted in PBS containing 1% BSA). After three washes with PBS (5 minutes each), sections were incubated with the secondary antibody for 30 minutes at room temperature: either a lissamine rhodamine B sulfonyl chloride-conjugated goat anti-rabbit antibody (5 μg/ml) or a fluorescein isothiocyanate-conjugated goat anti-mouse antibody (5 μg/ml). Finally, the sections were washed three times with PBS (10 minutes each wash). Slides were mounted with Slow-Fade anti-fade reagent (Molecular Probes, Inc., Eugene, OR) and observed under an Olympus IX 70 inverted microscope. Skeletal muscle tissues were harvested, minced with a scissors, homogenized in a Polytron tissue grinder for 30 seconds at a medium-range speed, and solubilized in a buffer containing 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, and 60 mmol/L octyl glucoside for 45 minutes at 4°C. Samples were centrifuged at 13,000 × g for 10 minutes at 4°C to remove insoluble debris. Soluble proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide) and transferred to nitrocellulose membranes. Blots were blocked for 1 hour in Tris-buffered saline Tween (TBST) (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.2% Tween 20) containing 4% powdered skim milk and 1% BSA. Then, the membranes were incubated for 1 hour with a given primary antibody (or an overnight incubation with the anti-dystrophin antibody), and diluted in TBST/1%BSA. After three washes with TBST, the blots were incubated for 30 minutes with horseradish peroxidase (HRP)-conjugated secondary antibodies, diluted in TBST/1%BSA. Antibody-bound proteins were detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). Skeletal muscle frozen sections were subjected to hematoxylin and eosin (H&E) staining, essentially as described.26Mikel UV Advanced Laboratory Methods in Histology and Pathology. Armed Forced Institute of Pathology/American Registry of Pathology, Washington, DC1994Google Scholar To detect damaged muscle fibers, EBD (stock solution: 10 mg/ml in PBS) was injected (0.1 ml/10 g body weight) into the right side of the peritoneal cavity, as previously described.27Hamer PW McGeachie JM Davies MJ Grounds MD Evans blue dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.J Anat. 2002; 200: 69-79Crossref PubMed Scopus (228) Google Scholar, 28Matsuda R Nishikawa A Tanaka H Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle.J Biochem (Tokyo). 1995; 118: 959-964Crossref PubMed Scopus (284) Google Scholar Then, animals were returned to their cages and were sacrificed 20 hours postinjection. Diaphragm and gastrocnemius muscles were dissected. After isolation, diaphragms were rinsed in PBS, fixed in 10% formalin and evaluated macroscopically. Gastrocnemius frozen sections were simply mounted with Slow-Fade anti-fade reagent (Molecular Probes, Inc.), and observed under an Olympus IX 70 inverted microscope to detect the EBD red autofluorescence signal. Calpain activity was assayed using a fluorimetric assay (Oncogene Research Products), as per the manufacturer's instructions. The assay measures the ability of calpain to digest the synthetic substrate Suc-LLVY-AMC. Released free AMC was measured fluorometrically at an excitation of 360–380 nm and an emission of 440–460 nm. Frozen samples (25–40 mg) of gastrocnemius muscle, collected from untreated (PBS only) and MG-132-treated mdx mice, were quickly minced, immediately diluted in lysis buffer provided by the manufacturer, and homogenized on ice. Homogenates were centrifuged at 14,000 × g in a pre-cooled table-top microcentrifuge. After determination of the protein content using the BCA protein assay (Pierce), aliquots of the supernatants were incubated with the fluorogenic substrate. A fluorimeter (BMG Labtechnologies, Inc., Durham, NC) was used to measure the hydrolysis of peptides. Previous studies have shown that the expression of dystrophin and of dystrophin-associated proteins is absent or greatly reduced in skeletal muscles from mdx mice. The lack of dystrophin is thought to cause sarcolemmal instability, which may render the dystrophin-glycoprotein complex more susceptible to proteolytic degradation.14Kamper A Rodemann HP Alterations of protein degradation and 2-D protein pattern in muscle cells of mdx and DMD origin.Biochem Biophys Res Commun. 1992; 189: 1484-1490Crossref PubMed Scopus (15) Google Scholar However, the role of the ubiquitin-dependent proteasomal pathway in muscle fiber degeneration in dystrophin-deficient muscles remains controversial. We reasoned that inhibition of the proteasomal pathway should rescue the expression and subcellular localization of dystrophin-associated proteins. For this purpose, we treated mdx mice with the potent, reversible, and cell-permeable proteasomal inhibitor CBZ-leucyl-leucyl-leucinal (MG-132).23Galbiati F Volonté D Minetti C Bregman DB Lisanti MP Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation: treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3.J Biol Chem. 2000; 275: 37702-37711Crossref PubMed Scopus (88) Google Scholar, 29Tsubuki S Saito Y Tomioka M Ito H Kawashima S Differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine.J Biochem (Tokyo). 1996; 119: 572-576Crossref PubMed Scopus (236) Google Scholar We first performed localized treatment, by injecting MG-132 (at a final concentration of 20 μmol/L) into the gastrocnemius muscle of mdx mice. To visualize the injected muscle, we pre-mixed MG-132 with a blue dye (1% India Ink in PBS). The gastrocnemius muscle from the other hindlimb of each animal served as an internal control. After 24 hours, skeletal muscle tissues were harvested from untreated and MG-132-treated hindlimbs. Frozen skeletal muscle tissue sections were prepared and examined by immunofluorescence (Figure 1). Immunostaining was performed with antibodies directed against β-dystroglycan (Figure 1A), α-dystroglycan (Figure 1B), α-sarcoglycan (Figure 1C), dystrophin (Figure 1D), and nNOS (Figure 1E). As expected, all these proteins were absent or expressed at very low levels in skeletal muscle fibers from untreated mdx muscles (Figure 1, A to E, upper panels). Interestingly, treatment with the proteasomal inhibitor MG-132 could efficiently rescue the expression level and subcellular localization of β-dystroglycan, α-dystroglycan, α-sarcoglycan, and dystrophin. Similar results were obtained using other structurally related proteasome inhibitors, such as MG-115 (CBZ-leucyl-leucyl-norvalinal) and ALLN (N-acetyl-leucyl-leucyl-norleucinal). The lower panels of Figure 1, A to D, show that β-dystroglycan, α-dystroglycan, α-sarcoglycan, and dystrophin were all clearly detectable at the plasma membrane and partially in the cytoplasm of muscle fibers from MG-132-treated skeletal muscles. However, nNOS expression levels were only slightly increased after MG-132 treatment (Figure 1E, lower panel). To independently verify these observations, tissue lysates from untreated and MG-132-treated mdx gastrocnemius muscle were subjected to Western blot analysis with antibodies directed against β-dystroglycan, α-dystroglycan, α-sarcoglycan, dystrophin, and nNOS. Figure 2 demonstrates that the expression levels of β-dystroglycan, α-sarcoglycan, and dystrophin are greatly increased in MG-132-treated skeletal muscles, as compared with the untreated controls. Immunoblotting with the α-dystroglycan antibody shows that MG-132 treatment results in the specific augmentation of a band, that we believe to be the precursor form of α-dystroglycan (Figure 2A). The heavily-glycosylated mature form of α-dystroglycan does not appear to be increased in MG-132-treated skeletal muscles, versus the untreated muscles. Previous results have shown that the binding of β-dystroglycan to α-dystroglycan occurs independently of α-dystroglycan glycosylation.30Sciandra F Schneider M Giardina B Baumgartner S Petrucci TC Brancaccio A Identification of the β-dystroglycan binding epitope within the C-terminal region of α-dystroglycan.Eur J Biochem. 2001; 268: 4590-4597Crossref PubMed Scopus (38) Google Scholar In support of this notion, our current data show that unglycosylated α-dystroglycan binds β-dystroglycan, as both are properly targeted to the plasma membrane (Figure 1., Figure 2.). Consistent with what we observed by immunofluorescence analysis (Figure 1E), nNOS expression levels were only moderately increased in MG-132-treated skeletal muscle (Figure 2B). The observation that MG-132 treatment is able to rescue dystrophin expression in mdx skeletal muscle fibers is noteworthy itself (Figure 1., Figure 2.). Interestingly, dystrophin migrates as a band of approximately 115 kd, instead of 426 kd expected for the full-length dystrophin. It has been previously shown that the mdx mouse lacks dystrophin expression due to a premature stop codon in the dystrophin gene. As a result, the dystrophin protein is produced as a truncated protein9Sicinski P Geng Y Ryder-Cook AS Barnard EA Darlison MG Barnard PJ The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.Science. 1989; 244: 1578-1580Crossref PubMed Scopus (1017) Google Scholar which, however, retains the N-terminal domain and is expected to migrate at ∼120 kd, as we observe here. It is important to note that, in this study, to detect dystrophin expression, we used a monoclonal antibody raised against the amino-terminal domain of dystrophin (NCL-DYS3). However, proteins with a premature termination codon are usually subjected to degradation.31Brogna S Pre-mRNA processing: insights from nonsense.Curr Biol. 2001; 11: R838-R841Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 32Iborra FJ Jackson DA Cook PR Coupled transcription and translation within nuclei of mammal
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