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Conditional Activation of MET in Differentiated Skeletal Muscle Induces Atrophy

2006; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês

10.1074/jbc.m610916200

1083-351X

Tiziana Crepaldi, Francesca Bersani, Claudio Scuoppo, Paolo Accornero, Chiara Prunotto, Riccardo Taulli, Paolo E. Forni, Christian Leo, Roberto Chiarle, Jennifer Griffiths, David J. Glass, Carola Ponzetto,

Genetic Neurodegenerative Diseases

Skeletal muscle atrophy is a common debilitating feature of many systemic diseases, including cancer. Here we examined the effects of inducing expression of an oncogenic version of the Met receptor (Tpr-Met) in terminally differentiated skeletal muscle. A responder mouse containing the Tpr-Met oncogene and GFP (green fluorescent protein) as a reporter was crossed with a transactivator mouse expressing tTA under the control of the muscle creatine kinase promoter. Tpr-Met induction during fetal development and in young adult mice caused severe muscle wasting, with decreased fiber size and loss of myosin heavy chain protein. Concomitantly, in the Tpr-Met-expressing muscle the mRNA of the E3 ubiquitin ligases atrogin-1/MAFbx, MuRF1, and of the lysosomal protease cathepsin L, which are markers of skeletal muscle atrophy, was significantly increased. In the same muscles phosphorylation of the Met downstream effectors Akt, p38 MAPK, and IκBα was higher than in normal controls. Induction of Tpr-Met in differentiating satellite cells derived from the double transgenics caused aberrant cell fusion, protein loss, and myotube collapse. Increased phosphorylation of Met downstream effectors was also observed in the Tpr-Met-expressing myotubes cultures. Treatment of these cultures with either a proteasomal or a p38 inhibitor prevented Tpr-Met-mediated myotube breakdown, establishing accelerated protein degradation consequent to inappropriate activation of p38 as the major route for the Tpr-Met-induced muscle phenotype. Skeletal muscle atrophy is a common debilitating feature of many systemic diseases, including cancer. Here we examined the effects of inducing expression of an oncogenic version of the Met receptor (Tpr-Met) in terminally differentiated skeletal muscle. A responder mouse containing the Tpr-Met oncogene and GFP (green fluorescent protein) as a reporter was crossed with a transactivator mouse expressing tTA under the control of the muscle creatine kinase promoter. Tpr-Met induction during fetal development and in young adult mice caused severe muscle wasting, with decreased fiber size and loss of myosin heavy chain protein. Concomitantly, in the Tpr-Met-expressing muscle the mRNA of the E3 ubiquitin ligases atrogin-1/MAFbx, MuRF1, and of the lysosomal protease cathepsin L, which are markers of skeletal muscle atrophy, was significantly increased. In the same muscles phosphorylation of the Met downstream effectors Akt, p38 MAPK, and IκBα was higher than in normal controls. Induction of Tpr-Met in differentiating satellite cells derived from the double transgenics caused aberrant cell fusion, protein loss, and myotube collapse. Increased phosphorylation of Met downstream effectors was also observed in the Tpr-Met-expressing myotubes cultures. Treatment of these cultures with either a proteasomal or a p38 inhibitor prevented Tpr-Met-mediated myotube breakdown, establishing accelerated protein degradation consequent to inappropriate activation of p38 as the major route for the Tpr-Met-induced muscle phenotype. The Met receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), 4The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; RMS, rhabdomyosarcoma; tTA, tetracycline transactivator; GFP, green fluorescent protein; PBS, phosphate-buffered saline; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; DM, differentiation medium; GM, growth medium; Dox, doxycycline; MCK, muscle creatine kinase; MEK, MAPK kinase kinase; C-26, colon-26; FKHR, Forkhead related transcription factors; TRE, TetO7-responsive element; MyHC, myosin heavy chain; TAMRA, 6-carboxytetramethylrhodamine; 6FAM, 6-carboxyfluorescein.4The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; RMS, rhabdomyosarcoma; tTA, tetracycline transactivator; GFP, green fluorescent protein; PBS, phosphate-buffered saline; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; DM, differentiation medium; GM, growth medium; Dox, doxycycline; MCK, muscle creatine kinase; MEK, MAPK kinase kinase; C-26, colon-26; FKHR, Forkhead related transcription factors; TRE, TetO7-responsive element; MyHC, myosin heavy chain; TAMRA, 6-carboxytetramethylrhodamine; 6FAM, 6-carboxyfluorescein. play an important role in skeletal muscle biology. Mouse embryos homozygous for a null mutation of the met or the hgf/sf locus fail to form muscles in the limbs, in the diaphragm, and in some areas of the tongue (1Prunotto C. Crepaldi T. Forni P.E. Ieraci A. Kelly R.G. Tajbakhsh S. Buckingham M. Ponzetto C. Dev. Dyn. 2004; 231: 582-591Crossref PubMed Scopus (35) Google Scholar) because of the inability of myogenic precursors to migrate from the somites and to reach these sites (2Bladt F. Riethmacher D. Isenmann S. Aguzzi A. Birchmeier C. Nature. 1995; 376: 768-771Crossref PubMed Scopus (1087) Google Scholar, 3Maina F. Casagranda F. Audero E. Simeone A. Comoglio P.M. Klein R. Ponzetto C. Cell. 1996; 87: 531-542Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 4Schmidt C. Bladt F. Goedecke S. Brinkmann V. Zschiesche W. Sharpe M. Gherardi E. Birchmeier C. Nature. 1995; 373: 699-702Crossref PubMed Scopus (1222) Google Scholar). In addition, hypomorphic mouse mutants with attenuated Met signaling show strong reduction of secondary fibers due to impaired proliferation of fetal myoblasts (3Maina F. Casagranda F. Audero E. Simeone A. Comoglio P.M. Klein R. Ponzetto C. Cell. 1996; 87: 531-542Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar).Conditional mouse models with muscle-specific Met loss or gain of function have not yet been described. Thus, our knowledge of Met-HGF/SF function in mature mammalian skeletal muscle derives by and large from in vitro studies. The Met-HGF/SF ligand/receptor pair is a positive regulator of satellite cell activation, proliferation, and migration during muscle regeneration (5Bischoff R. Dev. Dyn. 1997; 208: 505-515Crossref PubMed Scopus (172) Google Scholar, 6Gal-Levi R. Leshem Y. Aoki S. Nakamura T. Halevy O. Biochim. Biophys. Acta. 1998; 1402: 39-51Crossref PubMed Scopus (138) Google Scholar, 7Tatsumi R. Anderson J.E. Nevoret C.J. Halevy O. Allen R.E. Dev. Biol. 1998; 194: 114-128Crossref PubMed Scopus (505) Google Scholar). Satellite cells lie in close apposition to the myofiber beneath the basal lamina. Upon muscle injury, HGF/SF is released from the extracellular matrix (8Tatsumi R. Allen R.E. Muscle Nerve. 2004; 30: 654-658Crossref PubMed Scopus (75) Google Scholar). Quiescent satellite cells express the Met receptor (9Cornelison D.D. Wold B.J. Dev. Biol. 1997; 191: 270-283Crossref PubMed Scopus (716) Google Scholar), and the released HGF/SF stimulates their entry into the cell cycle. Besides increasing their proliferation, HGF/SF promotes their migration to the site of injury, as shown by its in vitro chemotactic activity (5Bischoff R. Dev. Dyn. 1997; 208: 505-515Crossref PubMed Scopus (172) Google Scholar, 10Suzuki J. Yamazaki Y. Li G. Kaziro Y. Koide H. Mol. Cell. Biol. 2000; 20: 4658-4665Crossref PubMed Scopus (86) Google Scholar). After injury, satellite cells enter the cell cycle, proliferate, and eventually cease proliferation and fuse with each other and with adjacent fibers to form multinucleated myotubes, effectively replacing the damaged fibers. Terminal differentiation of skeletal muscle requires the irreversible withdrawal of myoblasts from the cell cycle coupled to the up-regulation of muscle-specific genes (11Olson E.N. Klein W.H. Genes Dev. 1994; 8: 1-8Crossref PubMed Scopus (606) Google Scholar). Met activation counteracts the exit of satellite cells from the cell cycle and delays their myogenic differentiation (6Gal-Levi R. Leshem Y. Aoki S. Nakamura T. Halevy O. Biochim. Biophys. Acta. 1998; 1402: 39-51Crossref PubMed Scopus (138) Google Scholar, 12Anastasi S. Giordano S. Sthandier O. Gambarotta G. Maione R. Comoglio P. Amati P. J. Cell Biol. 1997; 137: 1057-1068Crossref PubMed Scopus (154) Google Scholar, 13Leshem Y. Spicer D.B. Gal-Levi R. Halevy O. J. Cell. Physiol. 2000; 184: 101-109Crossref PubMed Scopus (63) Google Scholar, 14Miller K.J. Thaloor D. Matteson S. Pavlath G.K. Am. J. Physiol. Cell Physiol. 2000; 278: 174-181Crossref PubMed Google Scholar).Activation of the HGF/Met axis has been implicated in development of rhabdomyosarcoma (RMS), a soft tissue tumor deriving from skeletal muscle cells. Recently we showed that Met silencing by RNA interference significantly impairs cell replication, survival, invasiveness, and anchorage-independent growth of RMS cells (15Taulli R. Scuoppo C. Bersani F. Accornero P. Forni P.E. Miretti S. Grinza A. Allegra P. Schmitt-Ney M. Crepaldi T. Ponzetto C. Cancer Res. 2006; 66: 4742-4749Crossref PubMed Scopus (133) Google Scholar). We had previously shown that Met is up-regulated in vivo by the chimeric transcription factor PAX3-FKHR (16Relaix F. Polimeni M. Rocancourt D. Ponzetto C. Schafer B.W. Buckingham M. Genes Dev. 2003; 17: 2950-2965Crossref PubMed Scopus (132) Google Scholar), the pathogenetic marker for Alveolar RMS, and that cells from mutant mouse embryos expressing a signalingdead Met receptor are unable to form colonies in soft agar after PAX3-FKHR transduction (15Taulli R. Scuoppo C. Bersani F. Accornero P. Forni P.E. Miretti S. Grinza A. Allegra P. Schmitt-Ney M. Crepaldi T. Ponzetto C. Cancer Res. 2006; 66: 4742-4749Crossref PubMed Scopus (133) Google Scholar). Furthermore, transgenic mice ubiquitously expressing HGF/SF are predisposed to develop RMS (17Takayama H. LaRochelle W.J. Sharp R. Otsuka T. Kriebel P. Anver M. Aaronson S.A. Merlino G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 701-706Crossref PubMed Scopus (374) Google Scholar) and show a very high incidence of RMS when combined with Ink4a/Arf inactivation (18Sharp R. Recio J.A. Jhappan C. Otsuka T. Liu S. Yu Y. Liu W. Anver M. Navid F. Helman L.J. DePinho R.A. Merlino G. Nat. Med. 2002; 8: 1276-1280Crossref PubMed Scopus (135) Google Scholar). All the above evidences suggest that Met plays a critical role in rhabdomyosarcoma genesis. Recently, a conditional mouse model of alveolar RMS obtained by targeting PAX3-FKHR expression to differentiating skeletal muscle has led to the hypothesis that this tumor may derive from terminally differentiating myofibers (19Keller C. Arenkiel B.R. Coffin C.M. El Bardeesy N. DePinho R.A. Capecchi M.R. Genes Dev. 2004; 18: 2614-2626Crossref PubMed Scopus (246) Google Scholar). In this work to further explore this concept we adopted the tetracycline transactivator (tTA) system to induce expression of an oncogenic form of Met (Tpr-Met) in differentiating muscle fibers. In the Tpr-Met oncoprotein the N-terminal region of Tpr, which includes two strong dimerization motifs, is fused to the tyrosine kinase moiety of Met, which is, thus, constitutively active in the absence of the ligand (20Park M. Dean M. Cooper C.S. Schmidt M. O'Brien S.J. Blair D.G. Vande Woude G.F. Cell. 1986; 45: 895-904Abstract Full Text PDF PubMed Scopus (430) Google Scholar). Expression of Tpr-Met in differentiating muscle did not result in development of musculoskeletal tumors but, rather, caused dramatic muscle wasting concomitant with the induction of proteasomal and lysosomal proteolysis.EXPERIMENTAL PROCEDURESGeneration and Identification of Transgenic Mice—Mice harboring the MCK-tTA (skeletal muscle-specific) promoter construct were kindly donated by Dr. P. Plotz (21Nagaraju K. Raben N. Loeffler L. Parker T. Rochon P.J. Lee E. Danning C. Wada R. Thompson C. Bahtiyar G. Craft J. Hooft V.H. Plotz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9209-9214Crossref PubMed Scopus (242) Google Scholar) and kept in a C57BL/6 background. MCK-tTA heterozygotes were interbred, and homozygous mice were selected by Southern and dot blot. The Tpr-Met cDNA (22Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Z.P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (885) Google Scholar) was digested with EcoRI, and overhangs were blunted using T4 DNA polymerase and ligated into the PvuII site of pBI-GFP vector (Clontech) to generate the bidirectional Tpr-Met-TRE-GFP responder construct. The Tpr-Met-TRE-GFP construct was cut with AseI, and a 5.9-kilobase gel-purified fragment was microinjected into the fertilized eggs of FVB mice in the San Raffaele-Telethon Core Facility for Conditional Mutagenesis (Milan, Italy). Founder mice were identified by PCR analysis of genomic DNA prepared from tail biopsies. A total of seven transgenic founders were obtained upon two series of microinjections. Ear fibroblasts from all responders but one (#20) expressed GFP upon infection with LV-TA1 lentiviral vector carrying the tTA under the phosphoglycerate kinase promoter. The various founders showed strong GFP expression variability (#12 > #8 > #9 > #4 > #2 > #15), with the highest levels found in mouse #12. Lines were established from these founders, analyzed by Southern blots, and maintained in a FVB background. Two independent target lines segregated in the F1 progeny from founder #12 (#12.2 and #12.9) and were chosen for breeding to MCK-tTA transactivator mice. Mice were genotyped by PCR analysis of tail genomic DNA. The tTA transgene was identified with primers annealing to tTA cDNA, 5′-AAG TGA TTA ACA GCG CAT TAG AGC-3′ and 5′-TTC AAG GCC GAA TAA GAA GGC TGG-3′, resulting in a 547-bp fragment. The Tpr-Met-TRE-GFP responder transgene was identified with a primer annealing to exon 20 of Met, 5′-AGA GGA GCC CCT CCT TAT CC-3′, and the other to the β-globin poly(A) sequence downstream of the Tpr-Met transgene, 5′-GGT CCC CAA ACT CAC CCT GAA GTT CTC-3′, resulting in a 665-bp fragment. The transgene integration sites in different Tpr-Met-TRE-GFP responder lines were analyzed by Southern blot. Ten micrograms of mouse tail genomic DNA were digested with HindIII restriction enzymes and subjected to Southern blotting and hybridization using Hybond-N+ nylon membrane (Amersham Biosciences) and ULTRAhyb (Ambion). The 32P-labeled probe (Rediprime, Amersham Biosciences) was a 1.2-kilobase fragment of human c-Met cDNA (from nucleotides 3225 to 4409). The MCK-tTA transgene status was analyzed after EcoRI digestion of genomic DNA with a 32P-labeled 1.5-kilobase probe prepared from EcoRI-HindIII-digested pTEToff plasmid (Clontech). Undigested DNA was used for dot blot analysis with the same probe. Crosses were carried out between MCK-tTA homozygotes and Tpr-Met-TRE-GFP heterozygotes. Doxycycline (Sigma) was diluted in 3% sucrose in water to a final concentration of 200 μg/ml and supplied as drinking water with changes every 2-3 days. Transmission of both transgenes followed typical Mendelian inheritance patterns. In vivo GFP fluorescence was monitored by using a fluorescent light box, illuminated by blue light fiber optics and imaged by Olympus Camedia camera. Litters from double transgenic animals maintained without Dox (TprMet Ind. mice) were screened at P1; 26 of 26 TprMet Ind. mice derived from line #12.9 and 16 of 26 TprMet Ind. mice derived from line #12.2 were green fluorescent and died perinatally. The lower penetrance of the lethal phenotype in TprMet Ind. mice derived from line #12.2 as compared with 100% penetrance of line #12.9 is likely due to positional effect and variegated expression. All subsequent experiments were carried out on TprMet Ind. mice derived from line #12.9. For postnatal induction, doxycycline was administered to pregnant mothers throughout gestation and lactation, and the Dox was removed at weaning (p21). In vivo imaging of GFP expression was detected 2-3 weeks after induction. Animals developing massive atrophy (50% weight loss with respect to uninduced controls) within two months of age were analyzed. Samples of muscle derived from an experimental mouse model of cancer cachexia were kindly provided together with the controls by Dr. Paola Costelli. Briefly, BALB/c mice were injected subcutaneously with the colon carcinoma cell line C-26 (5 × 105 cells per mouse) according to Matsumoto et al. (23Matsumoto T. Fujimoto-Ouchi K. Tamura S. Tanaka Y. Ishitsuka H. Br. J. Cancer. 1999; 79: 764-769Crossref PubMed Scopus (23) Google Scholar), and the hindlimb muscles were recovered 12 days later for Western blot analysis. All animal procedures were approved by the Ethical Commission of the University of Torino, Italy, and by the Italian Ministry of Health.Ear Fibroblasts and Satellite Cells Cultures—Ear biopsies from transgenic founders were minced and digested with 1 μg/μl collagenase/dispase (Roche Applied Science) in Dulbecco's PBS supplemented with CaCl2 and MgCl2 (D-PBS; Sigma) for 120 min at 37 °C with shaking. The digestion was stopped by adding 1:3 fetal bovine serum (FBS) (Euroclone), and crushed tissue was plated in Dulbecco's modified Eagle's medium (Euroclone) plus 10% FBS in 24-well plates. Fibroblasts adhered to the bottom of the plate after 1 week. Cells were passaged every 3-4 days. Satellite cells were isolated from muscles of 17-day-old double transgenic mice (MCKtTA/Tpr-Met-TRE-GFP) kept in Dox from conception. After removal of skin, fat tissue, and bones, hindlimb muscles were digested with 1 μg/μl collagenase/dispase in D-PBS for 40-50 min at 37 °C with shaking. During the proteolytic digestion, tissues were occasionally fragmented by repeated pipetting. The digestion was stopped by adding 1:3 fetal bovine serum in D-PBS. The debris was removed by filtration through a 70-μm sterile filter, and cells were collected by centrifugation. Cells were then resuspended in complete GM growth medium (F-10 HAM; Sigma) containing 20% fetal bovine serum, 3% chicken embryo extract and 2.5 ng/ml bovine fibroblast growth factor (Peprotech)). Cells were preplated overnight to discard contaminating fibroblasts and then non-adherent cells were plated in GM on collagen (0.1 mg/ml, Sigma)-coated plates. Single satellite cells started to be visible after 2-3 days of culture. Cells were passaged every 3 days, when they were ∼70% confluent using EDTA 0.5 mm in PBS for detachment. The cellular population underwent a proliferation crisis after 2-3 weeks, from which immortalized satellite cells arose. Under such conditions purity of satellite cells exceeded 99%. Proliferating cells were cultured for 20-25 passages at maximum. To obtain differentiation into myotubes, cells were plated at subconfluence on gelatin (0.5%, Sigma)-coated plates, maintained in GM for 24 h, and then shifted to DM differentiation medium (Dulbecco's modified Eagle's medium containing 5% horse serum (Euroclone)). Incubation was performed at 37 °C in a 5% CO2, water-saturated atmosphere, and all media were supplemented with 2 mml-glutamine, 100 units of penicillin and 0.1 mg/ml streptomycin.Lentiviral Vector Production and in Vitro Transduction—High titer lentiviral vector stock was produced in 293T cells by calcium phosphate-mediated transient transfection of the modified transfer and packaging vectors pMDLg/pRRE, pRSV-Rev, and pMD2.VSVG (24Vigna E. Cavalieri S. Ailles L. Geuna M. Loew R. Bujard H. Naldini L. Mol. Ther. 2002; 5: 252-261Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The viral p24 antigen concentration was determined by human immunodeficiency virus-1 p24 core profile enzyme-linked immunosorbent assay (PerkinElmer Life Sciences). Ear fibroblasts (1 × 105 cells in 35-mm diameter culture dishes) from Tpr-Met responder mice were transduced with 70 ng of p24 of lentiviral vector in presence or absence of doxycycline (1 μg/ml) and Polybrene (8 μg/ml).Inhibitors, Reagents, and Antibodies—PD98059 (20 μm), SB203580 (5-20 μm), wortmannin (100 nm), and MG132 (5 μm) were added after 24 h from DM shift. All inhibitors were purchased from Calbiochem. All reagents used were from FlukaChemie and Sigma. The following antibodies were used: anti-human Met (Santa Cruz, #sc-10); anti-mouse Met (Santa Cruz, #Sc-8057; Zymed Laboratories Inc., #3D4); anti-phospho-Erk-1,2 MAPK (Sigma, #M8159); anti-phospho-Akt (Ser-473; Cell Signaling, #9271S); anti-phospho-p38 MAPK (Cell Signaling; #4631); anti-α-tubulin (Sigma; #T5168); anti-desmin (Dako; #M0724); anti-GFP (Molecular Probes, #A-11122); anti-phospho-IκBα (Ser-32; Cell Signaling, #9241S); anti-Ki67 (Immunotech, #IM1920). Anti-myosin heavy chain (MyHC) MoAb (MF20) was kindly provided by Dr. M. Prat. Horseradish peroxidase-conjugated goat anti-rabbit, and rabbit anti-mouse antibodies were from Pierce.Western Blot Analysis—Myotube cultures and muscle tissues were lysed at room temperature in lysis buffer (50 mm Tris-HCl, pH 6.8, 10% glycerol, and 2% SDS) and sonicated. For phosphoprotein analysis, myotubes were lysed at 4 °C in EB buffer (20 mm Tris HCl, pH 7.4, 160 mm NaCl, 10% glycerol, 5 mm EDTA, pH 8, 1% Triton-X-100, protease inhibitor mixture, and 1 mm sodium orthovanadate). Protein concentration was determined by BCA assay (Pierce), and proteins were resolved in 10% SDS-PAGE gels and transferred to Hybond-C Extra nitrocellulose membranes (Amersham Biosciences). Immunoblots were developed with Super Signal West Pico chemiluminescent substrate (Pierce) and visualized on Amersham Biosciences Hyperfilm.Immunohistochemistry, Fiber Size Measurements, and Immunofluorescence—Either E20 embryos, P0 newborn mice, or dissected tibialis anterior muscle were fixed in 4% paraformaldehyde overnight, dehydrated, and embedded in paraffin. Sections for histological analysis (6-8 μm thick) were rehydrated and stained with hematoxylin-eosin. For immunohistochemistry, rehydrated sections were treated with 3% H2O2 and microwaved for 30 min in 10 mm Antigen Retrieval Citra (Biogenex). Sections were then incubated with primary antibody 1 h at room temperature. Incubation with biotinylated secondary antibody and peroxidase-conjugated streptavidin (both 15 min room temperature) were performed using the Dako LSAB 2 system peroxidase kit. Staining was developed by liquid diaminobenzidine chromogen (Biogenex) followed by hematoxylin counterstain. For double staining experiments, after Ki-67 peroxidase staining sections were decorated with MF-20 primary antibody followed by biotinylated secondary antibody and phosphatase-conjugated streptavidin using the same kit as above. Blue staining was developed by Fast Blu chromogen (Sigma). Fiber cross-sectional areas were measured using IMAGE J software (rsb.info.nih.gov/ij) at 40× magnification. GFP fluorescence was monitored in living myotubes. For indirect immunofluorescence studies, myotubes differentiated on 24-well plates were fixed with methanol/acetone and permeabilized with 0.5% Triton X-100, incubated with MF20 primary antibody and then with Cy3-conjugated anti-mouse antibody (Sigma). Nuclei were stained with 4′, 6-diamidino-2-phenylindole. Samples were viewed under a fluorescence-equipped inverted microscope (Leica). Pictures were taken with Evolution VF color cool camera and Image-Pro software.Quantitative Reverse Transcription-PCR—Total RNA was extracted using the Qiagen RNeasy midi kit. RNA integrity was checked using RNA 6000 Nano Assay kit and Agilent 2100 Bioanalyzer. Total RNA quantification was measured by PCR and fluorogenic 5′ nuclease assay (Taqman assay). cDNAs were synthesized using Taqman reverse transcription reagents kit (Applied Biosystems). For each RNA sample a control reaction without reverse transcriptase was also run to exclude genomic contamination. 25 ng of each sample was loaded in triplicate in the optical reaction plate. Taqman PCR Mastermix (Applied Biosystems), Taqman probe (200 nm), and primers targeting the gene of interest (900 nm) were added to each sample. A standard curve generated from known amounts of mouse genomic DNA was used to determine the absolute quantities of each RNA. The reactions were read using the ABI PRISM 7900HT sequence detection instrument. The results were normalized to endogenous control (cyclophilin). The following primers and probes sequences were used: atrogin-1 forward, 5′-GTGCTTACAACTGAACATCATGCA-3′; atrogin-1 reverse, 5′-TGGCCCAGGCTGACCA-3′; atrogin-1 probe, 6FAM-TCCCGCCCGTCACTCAGCCT-TAMRA; MuRF1 forward 5′-TGTCTGGAGGTCGTTTCCG-3′; MuRF1 reverse, 5′-TGCCGGTCCATGATCACTT-3′; MuRF1 probe, 6FAM-TGCCCCTCGTGCCGCCATAMRA; cathepsin L forward, 5′-TGACACAGGGTTCGTGGATATC-3′; cathepsin L reverse, 5′-CAGTCGCCACAGCCTTCA-3′; cathepsin L probe, 6FAM-AGCAAGAGAAAGCCCT-TAMRA; cyclophilin forward, 5′-CGTGGGCTCCGTCGTC-3′; cyclophilin reverse, 5′-CCCTTCTTCTTATCGTTGGCC-3′; cyclophilin probe, 6FAM-TTGCTGCCCGGACCCTCCG-TAMRA. All probes were synthesized by Applied Biosystems.Statistics—Data are expressed as the mean ± S.E. or ± S.D. as indicated. Differences between the induced mice and the control groups were tested by using the χ2 test. The level of significance was set at p < 0.05. A Pearson correlation coefficient was used to test the strength and relationship between variables.RESULTSGeneration of Bitransgenic Mice with Inducible Expression of Tpr-Met in Skeletal Muscle—To obtain transgenic mice in which expression of Tpr-Met can be specifically induced in muscle in a regulated manner, we adopted the Tet-on/off technology described by Gossen and Bujard (25Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar). The Tpr-Met-TRE-GFP responder construct was assembled by inserting the cDNAs of Tpr-Met and of the GFP reporter into the bidirectional plasmid pBI (Fig. 1A) (26Baron U. Freundlieb S. Gossen M. Bujard H. Nucleic Acids Res. 1995; 23: 3605-3606Crossref PubMed Scopus (265) Google Scholar). The responder construct was validated in cell transfection experiments (27Taulli R. Accornero P. Follenzi A. Mangano T. Morotti A. Scuoppo C. Forni P.E. Bersani F. Crepaldi T. Chiarle R. Naldini L. Ponzetto C. Cancer Gene Ther. 2005; 12: 456-463Crossref PubMed Scopus (33) Google Scholar) and then microinjected into fertilized mouse oocytes to generate responder transgenic mice (see "Experimental Procedures"). Bitransgenic mice (MCK-tTA/Tpr-Met-TRE-GFP; Fig. 1B) were obtained by crossing the T12 line (one of the highest responders) with the transgenic activator mouse MCKtTA, expressing tTA under the muscle creatine kinase (MCK) promoter (21Nagaraju K. Raben N. Loeffler L. Parker T. Rochon P.J. Lee E. Danning C. Wada R. Thompson C. Bahtiyar G. Craft J. Hooft V.H. Plotz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9209-9214Crossref PubMed Scopus (242) Google Scholar). The MCK promoter is active in mouse embryos from E14 onward (28Lyons G.E. Muhlebach S. Moser A. Masood R. Paterson B.M. Buckingham M.E. Perriard J.C. Development. 1991; 113: 1017-1029Crossref PubMed Google Scholar) when secondary myogenesis and terminal differentiation occur. Pregnant mothers were either given doxycycline (Dox) starting at the time of mating to block the responder transgene expression in the offspring (uninduced) or were left without treatment to induce its expression (Tpr-Met-induced). Fig. 1C shows E20 bitransgenic embryos, which developed in mothers kept with and without Dox (uninduced/Tpr-Met-induced). Induced bitransgenic embryos were immediately identifiable for their fluorescence and expressed Tpr-Met in their muscles (Fig. 1C, upper row and right panel). Dox administration prevented tTA induction keeping GFP and Tpr-Met at undetectable levels (Fig. 1C, lower row and right panel). The Tpr-Met-induced embryos had a characteristic "hunchback" appearance and lacked the S-shaped curvature of the vertebral column seen in control mice (Fig. 1C, middle panel). This is likely to be an indirect consequence of the strong reduction and disorganization of back muscles (Fig. 1D, left panel) that are required to support proper alignment of the vertebrae during development. As shown in Fig. 1D, middle and right panels, in Tpr-Met-induced mice the diaphragm was much thinner, and the tongue muscles were reduced with respect to controls. Induced newborn mice died perinatally, presumably due to respiratory and feeding defects.Induction of Tpr-Met in Skeletal Muscle Causes Muscle Atrophy—To better visualize the morphology of the muscle fibers, transverse sections of hindlimbs from newborn (P0) mice were stained with anti-desmin monoclonal antibody (Fig. 2A). This revealed reduced skeletal muscle masses and smaller fibers in Tpr-Met-induced mice as compared with uninduced controls. To quantitate muscle shrinkage we measured the cross-sectional areas of more than 300 fibers from Tpr-Met-induced and control mice (Fig. 2B). The mean fiber cross-sectional area of Tpr-Met-induced mice was reduced by 55% over the controls. Plotting the fiber area as frequency distributions showed a leftward shift, indicating an evident increase in the percentage of small fibers in the Tpr-Met-induced mice. To investigate the organization of individual fibers, longitudinal sections of hindlimbs were treated by Masson trichrome staining (Fig. 2C). The transverse pattern of Z-lines seen in control mice was barely detectable in Tpr-Met-induced mice, indicating that the fiber structure was highly disorganized. To verify the myosin content and the degree of proliferation, tongue transverse sections were doublestained with anti-MyHC monoclonal antibody and with anti-nuclear Ki67 rabbit antibody (Fig. 2D). In Tpr-Met-induced mice MyHC staining was decreased and was often absent in the central area of the fibers, suggesting myosin loss. The nuclei density was 10% higher in Tpr-Met induced with respect to control mice. The total number of Ki67-positive nuclei counted in whole tongue sections was similar in Tpr-Met-induced and control mice (12.100 + 350 versus 12.200 + 360), suggesting that the proliferation rate was not increased in Tpr-Met-induced mice and that the higher density of nuclei was due to muscle fiber shrinkage. Careful inspection of these sections did not yield any e