FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1990054118> ?p ?o ?g. }

• W1990054118 endingPage "35124" @default.
• W1990054118 startingPage "35113" @default.
• W1990054118 abstract "We have investigated the effect of tumor necrosis factor-α (TNF-α) on the production of extracellular matrix-degrading proteases in skeletal muscles. Using microarray, quantitative PCR, Western blotting, and zymography, we found that TNF-α drastically increases the production of matrix metalloproteinase (MMP)-9 from C2C12 myotubes. In vivo administration of TNF-α in mice increased the transcript level of MMP-9 in skeletal muscle tissues. Although TNF-α activated all the three MAPKs (i.e. ERK1/2, JNK, and p38), inhibition of ERK1/2 or p38 but not JNK blunted the TNF-α-induced production of MMP-9 from myotubes. Inhibition of Akt also inhibited the TNF-α-induced production of MMP-9. TNF-α increased the activation of transcription factors NF-κB and AP-1 but not SP-1 in myotubes. Overexpression of a dominant negative inhibitor of NF-κB or AP-1 blocked the TNF-α-induced expression of MMP-9 in myotubes. Similarly, point mutations in AP-1- or NF-κB-binding sites in MMP-9 promoter inhibited the TNF-α-induced expression of a reporter gene. TNF-α increased the activity of transforming growth factor-β-activating kinase-1 (TAK1). Furthermore, overexpression of a dominant negative mutant of TAK1 blocked the TNF-α-induced expression of MMP-9 and activation of NF-κB and AP-1. Our results also suggest that TNF-α induces MMP-9 expression in muscle cells through the recruitment of TRAF-2, Fas-associated protein with death domain, and TNF receptor-associated protein with death domain but not NIK or TRAF-6 proteins. We conclude that TAK1-mediated pathways are involved in TNF-α-induced MMP-9 production in skeletal muscle cells. We have investigated the effect of tumor necrosis factor-α (TNF-α) on the production of extracellular matrix-degrading proteases in skeletal muscles. Using microarray, quantitative PCR, Western blotting, and zymography, we found that TNF-α drastically increases the production of matrix metalloproteinase (MMP)-9 from C2C12 myotubes. In vivo administration of TNF-α in mice increased the transcript level of MMP-9 in skeletal muscle tissues. Although TNF-α activated all the three MAPKs (i.e. ERK1/2, JNK, and p38), inhibition of ERK1/2 or p38 but not JNK blunted the TNF-α-induced production of MMP-9 from myotubes. Inhibition of Akt also inhibited the TNF-α-induced production of MMP-9. TNF-α increased the activation of transcription factors NF-κB and AP-1 but not SP-1 in myotubes. Overexpression of a dominant negative inhibitor of NF-κB or AP-1 blocked the TNF-α-induced expression of MMP-9 in myotubes. Similarly, point mutations in AP-1- or NF-κB-binding sites in MMP-9 promoter inhibited the TNF-α-induced expression of a reporter gene. TNF-α increased the activity of transforming growth factor-β-activating kinase-1 (TAK1). Furthermore, overexpression of a dominant negative mutant of TAK1 blocked the TNF-α-induced expression of MMP-9 and activation of NF-κB and AP-1. Our results also suggest that TNF-α induces MMP-9 expression in muscle cells through the recruitment of TRAF-2, Fas-associated protein with death domain, and TNF receptor-associated protein with death domain but not NIK or TRAF-6 proteins. We conclude that TAK1-mediated pathways are involved in TNF-α-induced MMP-9 production in skeletal muscle cells. Skeletal muscle wasting or atrophy is a debilitating complication of several conditions, including immobilization, zero gravity space travel, and many chronic diseases such as cancer, heart failure, diabetes, AIDS, and sepsis (1Jackman R.W. Kandarian S.C. Am. J. Physiol. 2004; 287: C834-C843Crossref PubMed Scopus (704) Google Scholar, 2Kandarian S.C. Jackman R.W. Muscle Nerve. 2006; 33: 155-165Crossref PubMed Scopus (285) Google Scholar). In vivo, skeletal muscle fibers are surrounded by a layer of extracellular matrix (ECM) 3The abbreviations used are: ECM, extracellular matrix; DM, differentiation medium; DN, dominant negative; EMSA, electrophoretic mobility shift assay; ERK1/2, extracellular signal-related kinase; FADD, Fas-associated protein with death domain; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; NIK, NF-κB inducing kinase; PI3K, phosphoinositide 3-kinase; SEAP, secreted alkaline phosphatase; TAK1, transforming growth factor-β-activated kinase 1; TIMP, tissue inhibitor of matrix metalloproteinase; TNF, tumor necrosis factor; TRAF, TNF-receptor associated factor; TRADD, TNF receptor-associated protein with death domain; DMEM, Dulbecco's modified Eagle's medium; QRT-PCR, quantitative real time-PCR; IKK, IκB kinase. material called the basement membrane, which is composed of an internal basal lamina directly linked to sarcolemma and an external fibrillar lamina (3Sanes J.R. J. Biol. Chem. 2003; 278: 12601-12604Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The main components of basal lamina are type IV collagen, laminin, and proteoglycans rich in heparin sulfate. Genetic studies of muscular dystrophy patients and animal models of muscular dystrophy have provided strong evidence that the components of ECM are critical for maintaining structure and normal function of skeletal muscle (4Michele D.E. Campbell K.P. J. Biol. Chem. 2003; 278: 15457-15460Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 5Mayer U. J. Biol. Chem. 2003; 278: 14587-14590Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 6Helbling-Leclerc A. Zhang X. Topaloglu H. Cruaud C. Tesson F. Weissenbach J. Tome F.M. Schwartz K. Fardeau M. Tryggvason K. Guicheney P. Nat. Genet. 1995; 11: 216-218Crossref PubMed Scopus (568) Google Scholar, 7Jobsis G.J. Keizers H. Vreijling J.P. de Visser M. Speer M.C. Wolterman R.A. Baas F. Bolhuis P.A. Nat. Genet. 1996; 14: 113-115Crossref PubMed Scopus (217) Google Scholar). ECM is not only required to maintain tensile strength and elasticity in skeletal muscle tissues, it is also important for the assembly of neuromuscular and myotendinous junctions and for the regeneration of myofibers after injury or excessive exercise (3Sanes J.R. J. Biol. Chem. 2003; 278: 12601-12604Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Thus, the degradation of ECM would result in structural and functional deterioration and loss of skeletal muscle fibers. Matrix metalloproteinases (MMPs) are a family of calcium- and zinc-dependent proteolytic enzymes that function mainly in the extracellular matrix (8Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3890) Google Scholar). Genetic and pharmacological studies have provided compelling evidence that although MMPs are required for normal development and functioning, excessive production of MMPs leads to pathology of a wide range of tissues (9Vu T.H. Werb Z. Genes Dev. 2000; 14: 2123-2133Crossref PubMed Scopus (1052) Google Scholar). Accumulating evidence suggests that increased expression of MMPs could lead to loss of skeletal muscle mass in different physiological and pathophysiological conditions (10Carmeli E. Moas M. Reznick A.Z. Coleman R. Muscle Nerve. 2004; 29: 191-197Crossref PubMed Scopus (186) Google Scholar). Increased expression of MMP-2 and MMP-9 has been reported in the atrophic soleus and gastrocnemius muscles with a concomitant decrease in the levels of collagen I and collagen IV, two major ECM components present in skeletal muscle (11Giannelli G. De Marzo A. Marinosci F. Antonaci S. Histol. Histopathol. 2005; 20: 99-106PubMed Google Scholar, 12Reznick A.Z. Menashe O. Bar-Shai M. Coleman R. Carmeli E. Muscle Nerve. 2003; 27: 51-59Crossref PubMed Scopus (43) Google Scholar). MMP-2 and MMP-9 are strongly up-regulated in experimental ischemia/reperfusion injury leading to degradation of basal lamina in skeletal muscle (13Roach D.M. Fitridge R.A. Laws P.E. Millard S.H. Varelias A. Cowled P.A. Eur. J. Vasc. Endovasc. Surg. 2002; 23: 260-269Abstract Full Text PDF PubMed Scopus (102) Google Scholar). Furthermore, increased expression of MMPs has also been reported in atrophying skeletal muscle after nerve injury (14Kherif S. Dehaupas M. Lafuma C. Fardeau M. Alameddine H.S. Neuropathol. Appl. Neurobiol. 1998; 24: 309-319Crossref PubMed Scopus (84) Google Scholar), heart failure (15Schiotz Thorud H.M. Stranda A. Birkeland J.A. Lunde P.K. Sjaastad I. Kolset S.O. Sejersted O.M. Iversen P.O. Am. J. Physiol. 2005; 289: R389-R394Google Scholar, 16Carvalho R.F. Dariolli R. Justulin Junior L.A. Sugizaki M.M. Politi Okoshi M. Cicogna A.C. Felisbino S.L. Dal Pai-Silva M. Int. J. Exp. Pathol. 2006; 87: 437-443Crossref PubMed Scopus (32) Google Scholar), and in the pathogenesis of inflammatory myopathies (17Choi Y.C. Dalakas M.C. Neurology. 2000; 54: 65-71Crossref PubMed Google Scholar, 18Schoser B.G. Blottner D. Stuerenburg H.J. Acta Neurol. Scand. 2002; 105: 309-313Crossref PubMed Scopus (47) Google Scholar, 19Kieseier B.C. Schneider C. Clements J.M. Gearing A.J. Gold R. Toyka K.V. Hartung H.P. Brain. 2001; 124: 341-351Crossref PubMed Scopus (90) Google Scholar). Several lines of evidence suggest that TNF-α plays a major role in the development of muscular abnormalities resulting in a loss of skeletal muscle mass and function (20Li Y.P. Reid M.B. Curr. Opin. Rheumatol. 2001; 13: 483-487Crossref PubMed Scopus (99) Google Scholar). Increased levels of TNF-α have been observed under conditions that lead to skeletal muscle atrophy such as cachexia induced by bacteria, human immunodeficiency virus, chronic heart failure, and cancer (21Spate U. Schulze P.C. Curr. Opin. Clin. Nutr. Metab. Care. 2004; 7: 265-269Crossref PubMed Scopus (149) Google Scholar). TNF-α transduces its biological activities by binding to two 55- and 75-kDa receptors (22Aggarwal B.B. Nat. Rev. Immunol. 2003; 3: 745-756Crossref PubMed Scopus (2164) Google Scholar, 23Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1376) Google Scholar). Trimeric occupation of TNF receptors by the ligand results in the recruitment of receptor-specific proteins and promotes the activation of transcription factors such as nuclear factor-κB (NF-κB) and AP-1 (activator protein-1) through activation of a cascade of upstream kinases, including IκB kinase (IKK), the MAPKs, and Akt (22Aggarwal B.B. Nat. Rev. Immunol. 2003; 3: 745-756Crossref PubMed Scopus (2164) Google Scholar, 24Chen G. Goeddel D.V. Science. 2002; 296: 1634-1635Crossref PubMed Scopus (1498) Google Scholar). Recent reports also suggest that transforming growth factor-β-activated kinase1 (TAK1), a member of the MAP3K family, is critical for TNF-α-induced activation of NF-κB and MAPK signaling pathways, especially p38 (25Adhikari A. Xu M. Chen Z.J. Oncogene. 2007; 26: 3214-3226Crossref PubMed Scopus (346) Google Scholar, 26Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 27Shim J.H. Xiao C. Paschal A.E. Bailey S.T. Rao P. Hayden M.S. Lee K.Y. Bussey C. Steckel M. Tanaka N. Yamada G. Akira S. Matsumoto K. Ghosh S. Genes Dev. 2005; 19: 2668-2681Crossref PubMed Scopus (593) Google Scholar). Although the precise role of various signaling proteins and transcription factors in TNF-α-induced skeletal muscle atrophy remains to be established, some of these molecules may serve as important molecular targets for the prevention of skeletal muscle loss in future therapies. Indeed, recent studies have shown that the inhibition of NF-κB, a transcription factor that is also activated by TNF-α, can prevent skeletal muscle loss in conditions known to exacerbate muscle-wasting (28Acharyya S. Villalta S.A. Bakkar N. Bupha-Intr T. Janssen P.M. Carathers M. Li Z.W. Beg A.A. Ghosh S. Sahenk Z. Weinstein M. Gardner K.L. Rafael-Fortney J.A. Karin M. Tidball J.G. Baldwin A.S. Guttridge D.C. J. Clin. Investig. 2007; 117: 889-901Crossref PubMed Scopus (359) Google Scholar, 29Cai D. Frantz J.D. Tawa Jr., N.E. Melendez P.A. Oh B.C. Lidov H.G. Hasselgren P.O. Frontera W.R. Lee J. Glass D.J. Shoelson S.E. Cell. 2004; 119: 285-298Abstract Full Text Full Text PDF PubMed Scopus (1084) Google Scholar, 30Mourkioti F. Kratsios P. Luedde T. Song Y.H. Delafontaine P. Adami R. Parente V. Bottinelli R. Pasparakis M. Rosenthal N. J. Clin. Investig. 2006; 116: 2945-2954Crossref PubMed Scopus (244) Google Scholar). We hypothesize that one of the mechanisms by which TNF-α can induce loss of skeletal muscle mass and function is through augmenting the production of ECM-degrading enzymes from skeletal muscle cells. To test this hypothesis, we have performed a genome-wide microarray analysis using RNA isolated from control and TNF-α-treated cultured C2C12 myotubes. Among others, we have identified MMP-9, a type IV collagenase, as one of the highly up-regulated ECM proteases in TNF-α-treated C2C12 myotubes. In this study, for the first time we demonstrate that TNF-α causes spurious expression and production of MMP-9 from skeletal muscle cells both in vitro and in vivo. We have also delineated the molecular pathways that lead to enhanced expression of MMP-9 in C2C12 myotubes in response to TNF-α treatment. Our data suggest that TAK1, ERK1/2, p38 MAPK, NF-κB, and AP-1 transcription factors constitute the biochemical signaling pathway(s) that leads to the enhanced production of MMP-9 in C2C12 myotubes in response to TNF-α. Materials—Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were obtained from Invitrogen. Horse serum was purchased from Sigma. LY294002, wortmannin, PD98059, SP600125, and SB203580 were obtained from Calbiochem. Poly(dI·dC) was from GE Healthcare. [γ-32P]ATP (specific activity, 3000 (111 TBq) Ci/mmol) was obtained from MP Biomedicals (Solon, OH). Protein A-Sepharose beads were obtained from Pierce. Recombinant mouse TNF-α protein was from BioVision (Mountain View, CA). Effectene transfection reagent was obtained from Qiagen (Valencia, CA). NF-κB consensus oligonucleotides and luciferase assay kits were purchased from Promega (Madison, WI). Phospho-specific rabbit polyclonal anti-p44/42 (Thr-202/Tyr-204), anti-p38 (Thr-180/Tyr-182), anti-Akt (Ser-473), anti-IκBα (Ser-32), and anti-TAK1 (Ser-412) were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against JNK1, p50, p65, c-Rel, Bcl3, TAK1, and c-Jun proteins and AP-1 and SP-1 consensus oligonucleotides were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against mouse MMP-9 and MMP-2 proteins were obtained from R & D Systems (Minneapolis, MN). Mice—C57BL6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed and fed in stainless steel cages on a 10-h on and 14-h off lighting schedule. Prior approval was obtained from the Institutional Animal Care and Use Committee of the Jerry L. Pettis Memorial VA Medical Center, Loma Linda, CA, for conducting experiments with mice. All procedures were conducted in strict accordance with the Public Health Service and Department of Veterans Affair animal welfare policy. Cell Culture—C2C12 and L6 myoblastic cell lines were obtained from the American Type Culture Collection (Manassas, VA). These cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. C2C12 myoblasts were differentiated into myotubes by incubation in differentiation medium (DM, 2% horse serum in DMEM) for 96 h as described (31Dogra C. Changotra H. Mohan S. Kumar A. J. Biol. Chem. 2006; 281: 10327-10336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 32Dogra C. Changotra H. Wedhas N. Qin X. Wergedal J.E. Kumar A. FASEB J. 2007; 21: 1857-1869Crossref PubMed Scopus (188) Google Scholar). Myotubes were maintained in DM, and medium was changed every 48 h. L6 myoblasts were differentiated into myotubes by incubation in 0.5% fetal bovine serum for 48–72 h. Plasmid Constructs—Full-length mouse c-Jun was isolated from cDNA made from C2C12 myoblasts. Briefly, total RNA was isolated, and first strand cDNA was generated using oligo(dT) primer (from Ambion) and OmniScript reverse transcriptase according to the manufacturer's instructions (Qiagen). c-Jun construct was prepared by amplifying the murine c-Jun cDNA (GenBank™ accession number NM_010591) with the following primers: sense 5′-CGT TCT ATG ACT GCA AAG ATG G-3′ (c-Jun forward), and antisense, 5′-CAG CCC TGA CAG TCT GTT CT-3′ (c-Jun reverse). A dominant negative mutant of c-Jun, called TAM-67 (33Matthews C.P. Birkholz A.M. Baker A.R. Perella C.M. Beck Jr., G.R. Young M.R. Colburn N.H. Cancer Res. 2007; 67: 2430-2438Crossref PubMed Scopus (38) Google Scholar), was generated by removal of the transactivational domain (amino acids 3–122) of wild-type c-Jun by PCR using the following primers: sense 5′-ATG ACT AGC CAG AAC ACG CTT CCC A-3′ (TAM67 forward) and antisense, 5′-CAG CCC TGA CAG TCT GTT CT-3′ (c-Jun reverse). All PCR amplifications were performed using Pfu DNA polymerase (Stratagene). PCR products were cloned into pCR®Blunt II TOPO vector (Invitrogen), and the authenticity of cDNA was confirmed by automated DNA sequencing. Finally, c-Jun or TAM67 cDNA was excised from pCR®Blunt II TOPO vector using HindIII and XbaI restriction enzymes and inserted into pcDNA3 plasmid (Invitrogen) at HindIII and XbaI sites. The expression of proteins from plasmid constructs was confirmed using T7 based on the in vitro translation kit (Promega). The pcDNA3-FLAG-IκBαΔN plasmid has been described in one of our recent publications (34Kumar A. Murphy R. Robinson P. Wei L. Boriek A.M. FASEB J. 2004; 18: 1524-1535Crossref PubMed Scopus (99) Google Scholar). Plasmid constructs encoding murine wild-type HA-TAK1 or a dominant negative mutant (HA-TAK1K63W) and His-MKK6 as described (35Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1019) Google Scholar) were kindly provided by Dr. Jun Ninomiya-Tsuji, North Carolina State University. The 0.67-kb fragment of the MMP-9 promoter construct, together with two deletions (-599 and -73) and three mutation constructs (AP-1 mutant, TGAGTCA to TTTGTCA; NF-κB mutant, GGAATTCCCC to TTAATTCCCC; Sp-1 mutant, CCGCCC to CCAACC) ligated to the firefly luciferase reporter gene (36Han S. Ritzenthaler J.D. Sitaraman S.V. Roman J. J. Biol. Chem. 2006; 281: 29614-29624Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) were a gift from Dr. ShouWei Han, of Emory University School of Medicine. Dominant negative constructs for TRADD, FADD, TRAF2, TRAF6, and NIK were kindly provided by Prof. Bharat B. Aggarwal of the University of Texas M. D. Anderson Cancer Center. pTAL-SEAP and pNF-κB-SEAP plasmids were purchased from Clontech. Transient Transfection and Reporter Assays—C2C12 myoblasts plated in either 6-well or 24-well tissue culture plates were transfected with different plasmids using Effectene transfection reagent according to the protocol suggested by the manufacturer (Qiagen). Transfection efficiency was controlled by cotransfection of myoblasts with pSV-β-galactosidase plasmid. When >90% confluent, the cells were differentiated by changing medium to DM. After appropriate treatments, specimens were processed for luciferase and β-galactosidase expression using the luciferase and β-galactosidase assay systems with reporter lysis buffer per the manufacturer’s instructions (Promega, Madison, WI). Luciferase measurements were made using a luminometer (model 3010, Analytic Scientific Instrumentation). Alkaline phosphatase activity in culture supernatants was measured by a standard assay using para-nitrophenyl phosphate as a substrate. Gelatin Zymography—C2C12 myoblasts were differentiated into myotubes by incubation in DM for 96 h. Myotubes were treated with TNF-α for 14 h in serum-free medium, and the conditioned medium collected was subjected to gelatin zymography as described (37Kumar A. Dhawan S. Mukhopadhyay A. Aggarwal B.B. FEBS Lett. 1999; 462: 140-144Crossref PubMed Scopus (63) Google Scholar). Unconditioned medium was used as a negative control. In brief, conditioned media samples were separated on 10% SDS-polyacrylamide gels containing 1 mg/ml gelatin B (Fisher) under nonreducing conditions. Gels were washed in 2.5% Triton X-100 for 1 h at room temperature followed by incubation in reaction buffer (50 mm Tris-HCl (pH 7.5), 50 mm NaCl, 5 mm CaCl2, and 0.02% sodium azide) for 16 h at 37 °C. To visualize gelatinolytic bands, gels were stained with Coomassie Blue at room temperature followed by extensive washing in destaining buffer (10% methanol and 10% acetic acid in distilled water). The gels were dried and scanned under a densitometer for determination of gelatinolytic activity. Kinase Assays—The detailed protocol for assaying the JNK1 and IKK has been described previously (31Dogra C. Changotra H. Mohan S. Kumar A. J. Biol. Chem. 2006; 281: 10327-10336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 38Kumar A. Chaudhry I. Reid M.B. Boriek A.M. J. Biol. Chem. 2002; 277: 46493-46503Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). For measuring the TAK1 activity, 800 μg of protein was immunoprecipitated using rabbit polyclonal TAK1 antibody (Santa Cruz Biotechnology, Inc.), and the immune complex was collected using protein A-Sepharose beads. After washing two times with lysis buffer and two times with kinase buffer (50 mm HEPES (pH 7.4), 10 mm MgCl2, and 1 mm dithiothreitol), the beads were suspended in 20 μl of kinase assay mixture containing 50 mm HEPES (pH 7.4), 20 mm MgCl2, 2 mm dithiothreitol, 10 μCi of [γ-32P]ATP, 1 μm unlabeled ATP, and 2 μg of His-MKK6 as substrate. After incubation at 37 °C for 15 min, the reaction was terminated by boiling with 20 μl of 2× Laemmli sample buffer for 3 min. Finally, the protein was resolved on 10% polyacrylamide gel; the gel was dried, and the radioactive bands were visualized by exposing to a PhosphorImager screen and quantified using ImageQuant software (GE Healthcare). Western Blotting—Western blot was performed to measure the levels of different proteins using a standard protocol as described previously (32Dogra C. Changotra H. Wedhas N. Qin X. Wergedal J.E. Kumar A. FASEB J. 2007; 21: 1857-1869Crossref PubMed Scopus (188) Google Scholar, 39Dogra C. Hall S.L. Wedhas N. Linkhart T.A. Kumar A. J. Biol. Chem. 2007; 282: 15000-15010Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The dilution for all the primary antibodies was 1:100. Electrophoretic Mobility Shift Assay (EMSA)—The activation of NF-κB, AP-1, and SP-1 transcription factors was measured by EMSA. The detailed method for preparation of nuclear and cytoplasmic extracts and EMSA has been described in detail in our recent publications (31Dogra C. Changotra H. Mohan S. Kumar A. J. Biol. Chem. 2006; 281: 10327-10336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 40Kumar A. Boriek A.M. FASEB J. 2003; 17: 386-396Crossref PubMed Scopus (229) Google Scholar). Gene Expression Analysis Using Microarray Techniques—Total RNA was isolated from C2C12 myotubes 18 h after TNF-α treatment, using the Agilent total RNA isolation kit (Agilent Technologies, Palo Alto, CA). Each experiment was performed with a minimum of five replicates. The total RNA concentration was determined by NanoDrop spectrophotometer, and RNA quality was determined by 18 S/28 S ribosomal peak intensity on an Agilent Bioanalyzer. For microarray expression profiling and real time PCR, RNA samples were used only if they showed little to no degradation. Custom cDNA slides were spotted with Oligator “MEEBO” mouse genome set with 38,467 cDNA probes (Illumina, Inc., San Diego), which allows interrogation of 25,000 genes. A Q-Array2 robot (Genetix) was used for spotting. The array includes positive controls, doped sequences, and random sequence to ensure correct gene expression values were obtained from each array. A total of 250 ng of RNA was used to synthesize double-stranded cDNA using the low RNA input fluorescent linear application kit (Agilent). The microarray slides were scanned using a GSI Lumonics ScanArray 4200A Genepix scanner (Axon). The image intensities were analyzed using the ImaGene 5.6 software (Biodiscovery, Inc., El Segundo, CA). Expression analysis of microarray experiments was performed with GeneSpring 7.1 (Silicon Genetics, Palo Alto, CA) using the raw intensity data generated by the ImaGene software. Local background-subtracted total signal intensities were used as intensity measures, and the data were normalized using per spot and per chip LOWESS normalization. The changes in transcript levels were analyzed utilizing a t test with Benjamini and Hochberg Multiple Testing Correction. RNA Isolation and Quantitative Real Time-PCR—RNA isolation and QRT-PCR was performed to measure the mRNA level of different MMPs and TIMPs following a method described recently (31Dogra C. Changotra H. Mohan S. Kumar A. J. Biol. Chem. 2006; 281: 10327-10336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 32Dogra C. Changotra H. Wedhas N. Qin X. Wergedal J.E. Kumar A. FASEB J. 2007; 21: 1857-1869Crossref PubMed Scopus (188) Google Scholar). The sequence of the primers was as follows: MMP-2, 5′-ACA GCC AGA GAC CTC AGG GT-3′ (forward) and 5′-CAG CAC AGG ACG CAG AGA AC-3′ (reverse); MMP-9, 5′-GCG TGT CTG GAG ATT CGA CTT G-3′ (forward) and CAT GGT CCA CCT TGT TCA CCT C (reverse); MMP-14, 5′-ATT TGC TGA GGG TTT CCA CG-3′ (forward) and 5′-TCG GCA GAA TCA AAG TGG GT-3′ (reverse); TIMP-1, 5′-TTG CAT CTC TGG CAT CTG GCA T-3′ (forward) and 5′-GAT ATC TGC GGC ATT TCC CAC A-3′ (reverse); TIMP-2, 5′-GTGACTTCATTGTGCCCTGGG-3′ (forward) and 5′-TGG GAC AGC GAG TGA TCT TGC-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5′-ATG ACA ATG AAT ACG GCT ACA GCA A-3′ (forward) and 5′-GCA GCG AAC TTT ATT GAT GGT ATT-3′ (reverse). Data normalization was accomplished using the endogenous control gene glyceraldehyde-3-phosphate dehydrogenase, and the normalized values were subjected to a 2−ΔΔCt formula to calculate the fold change between the control and experiment groups. The formula and its derivations were obtained from the ABI Prism 7900 sequence detection system user guide. Statistical Analysis—Results are expressed as mean ± S.D. The Student’s t test or analysis of variance was used to compare quantitative data populations with normal distributions and equal variance. A value of p < 0.05 was considered statistically significant unless otherwise specified. Here we have investigated the effects of TNF-α on the expression of ECM-degrading proteases, especially MMPs in C2C12 myotubes. We have also delineated the molecular pathways that lead to increased expression of MMP-9 in response to TNF-α in C2C12 myotubes. The concentrations of TNF-α and various pharmacological inhibitors used in this study are within physiological range and have been widely used in different cell types, including skeletal muscle. TNF-α Modulates the Expression of MMPs and Their Inhibitors in Cultured Myotubes—We employed a microarray approach to determine how TNF-α affects the expression of various MMPs and their inhibitors in cultured C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes by incubation in differentiation medium for 96 h. The myotubes were then treated with recombinant mouse TNF-α protein (10 ng/ml) for 18 h. The total RNA isolated from control or TNF-α-treated myotubes was subjected to microarray analysis for the expression of MMP family genes. Interestingly, among 40 genes of the MMP family present in our microarray slides (supplemental Table 1S), the expression of only a few MMPs and TIMPs was significantly altered in TNF-treated myotubes compared with controls (Table 1). In particular, the expression of MMP-9, MMP-14, and TIMP-1 was significantly increased, whereas the expression of TIMP-2 and MMP-2 was reduced.TABLE 1List of matrix metalloproteinase-related genes significantly affected in TNF-treated myotubesGene name-Fold changep valueDescriptionTimp11.6<0.001Tissue inhibitor of metalloproteinase 1Mmp91.50.006Matrix metalloproteinase 9Mmp141.10.010Matrix metalloproteinase 14Mmp20.90.003Matrix metalloproteinase 2Timp20.80.027Tissue inhibitor of metalloproteinase 2 Open table in a new tab To validate the microarray data for the expression of MMPs and TIMPs, we performed QRT-PCR using their specific primers and RNA samples used in the microarray experiments. Interestingly, the mRNA level of MMP-9 was found to be drastically higher (>8-fold) in TNF-α-treated myotubes compared with untreated control myotubes (Fig. 1A). Indeed, in several other independent experiments, we found that the mRNA level of MMP-9 was increased up to 50-fold within 24 h of treatment of myotubes with TNF-α (data not shown). On the other hand, the increase in the mRNA level of TIMP-1, an inhibitor of MMP-9, was only ∼2-fold in QRT-PCR (Fig. 1A). Furthermore, QRT-PCR analysis also confirmed that the changes in the mRNA levels of MMP-2, MMP-14, and TIMP-2 were significant but not drastic (Fig. 1A). These data provide the first evidence that TNF-α can affect the expression of various MMP-related genes in C2C12 myotubes. TNF-α Augments the Production of MMP-9 in Skeletal Muscle Cells Both in Vitro and in Vivo—Because MMP-9 is one of the major ECM-degrading enzymes (8Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3890) Google Scholar) and its transcript level was increased on treatment of myotubes with TNF-α, we investigated whether the increased expression of MMP-9 was associated with increased secretion of MMP-9 in culture supernatants. C2C12 myotubes were incubated in serum-free medium with increasing amounts of TNF-α for 14 h, and the expression of MMP-9 was studi" @default.
• W1990054118 created "2016-06-24" @default.
• W1990054118 creator A5002997713 @default.
• W1990054118 creator A5008901932 @default.
• W1990054118 creator A5029876277 @default.
• W1990054118 creator A5058532609 @default.
• W1990054118 creator A5070799507 @default.
• W1990054118 creator A5074350735 @default.
• W1990054118 creator A5078802338 @default.
• W1990054118 date "2007-11-01" @default.
• W1990054118 modified "2023-10-17" @default.
• W1990054118 title "Tumor Necrosis Factor-α Augments Matrix Metalloproteinase-9 Production in Skeletal Muscle Cells through the Activation of Transforming Growth Factor-β-activated Kinase 1 (TAK1)-dependent Signaling Pathway" @default.
• W1990054118 cites W1259600258 @default.
• W1990054118 cites W1501881225 @default.
• W1990054118 cites W1527845641 @default.
• W1990054118 cites W1548219170 @default.
• W1990054118 cites W1575383081 @default.
• W1990054118 cites W1583835478 @default.
• W1990054118 cites W1600472471 @default.
• W1990054118 cites W1876517475 @default.
• W1990054118 cites W1975615803 @default.
• W1990054118 cites W1985266648 @default.
• W1990054118 cites W1985534077 @default.
• W1990054118 cites W1990877246 @default.
• W1990054118 cites W1992744008 @default.
• W1990054118 cites W1994787439 @default.
• W1990054118 cites W2006311087 @default.
• W1990054118 cites W2011211995 @default.
• W1990054118 cites W2015177938 @default.
• W1990054118 cites W2021356484 @default.
• W1990054118 cites W2025922826 @default.
• W1990054118 cites W2027086778 @default.
• W1990054118 cites W2027745468 @default.
• W1990054118 cites W2032948190 @default.
• W1990054118 cites W2038996595 @default.
• W1990054118 cites W2039645505 @default.
• W1990054118 cites W2039666900 @default.
• W1990054118 cites W2042087846 @default.
• W1990054118 cites W2053043355 @default.
• W1990054118 cites W2053761680 @default.
• W1990054118 cites W2055336582 @default.
• W1990054118 cites W2056222248 @default.
• W1990054118 cites W2059128799 @default.
• W1990054118 cites W2067680656 @default.
• W1990054118 cites W2069952825 @default.
• W1990054118 cites W2070782085 @default.
• W1990054118 cites W2073544673 @default.
• W1990054118 cites W2075303668 @default.
• W1990054118 cites W2075604614 @default.
• W1990054118 cites W2076277955 @default.
• W1990054118 cites W2079351435 @default.
• W1990054118 cites W2080727313 @default.
• W1990054118 cites W2094431347 @default.
• W1990054118 cites W2094835614 @default.
• W1990054118 cites W2095013915 @default.
• W1990054118 cites W2095245008 @default.
• W1990054118 cites W2098874403 @default.
• W1990054118 cites W2099918421 @default.
• W1990054118 cites W2100741744 @default.
• W1990054118 cites W2109213714 @default.
• W1990054118 cites W2112203191 @default.
• W1990054118 cites W2116551193 @default.
• W1990054118 cites W2124632579 @default.
• W1990054118 cites W2127808488 @default.
• W1990054118 cites W2135124706 @default.
• W1990054118 cites W2153141596 @default.
• W1990054118 cites W2153445234 @default.
• W1990054118 cites W2160824162 @default.
• W1990054118 cites W2170720109 @default.
• W1990054118 cites W2171726169 @default.
• W1990054118 cites W37880063 @default.
• W1990054118 cites W4250908540 @default.
• W1990054118 doi "https://doi.org/10.1074/jbc.m705329200" @default.
• W1990054118 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4154379" @default.
• W1990054118 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17897957" @default.
• W1990054118 hasPublicationYear "2007" @default.
• W1990054118 type Work @default.
• W1990054118 sameAs 1990054118 @default.
• W1990054118 citedByCount "58" @default.
• W1990054118 countsByYear W19900541182012 @default.
• W1990054118 countsByYear W19900541182013 @default.
• W1990054118 countsByYear W19900541182014 @default.
• W1990054118 countsByYear W19900541182016 @default.
• W1990054118 countsByYear W19900541182017 @default.
• W1990054118 countsByYear W19900541182020 @default.
• W1990054118 countsByYear W19900541182021 @default.
• W1990054118 countsByYear W19900541182022 @default.
• W1990054118 crossrefType "journal-article" @default.
• W1990054118 hasAuthorship W1990054118A5002997713 @default.
• W1990054118 hasAuthorship W1990054118A5008901932 @default.
• W1990054118 hasAuthorship W1990054118A5029876277 @default.
• W1990054118 hasAuthorship W1990054118A5058532609 @default.
• W1990054118 hasAuthorship W1990054118A5070799507 @default.
• W1990054118 hasAuthorship W1990054118A5074350735 @default.
• W1990054118 hasAuthorship W1990054118A5078802338 @default.
• W1990054118 hasBestOaLocation W19900541181 @default.
• W1990054118 hasConcept C109523444 @default.
• W1990054118 hasConcept C118131993 @default.

• next