Journal of Cell Science 1 Niu Airu TNF myogenesis activating of by activation mechanical the mediates Src Article Research K6(lokona A26 op8MP aha and Moreover, pathway 2007). MAPK al., release p38 et (Zhan to myoblasts differentiation MAP2K6) myogenic as ensuing stimulation, known (also mechanical MKK6 to related TNF the autocrine into response that, insights previously showed gain in we To in mechanotransduction, understood. of MAPK well mechanism loci p38 not myogenic activates is stimulation MPCs of mechanical regulation which through epigenetic Lluı 2009; of Puri, and as (Guasconi component expression gene key myogenic on a necessary turns MAPK a that 2001), ‘switch’ p38 Gardiner, sufficient and activates and Martineau 2001; stimulation al., mechanical et muscle (Boppart in that studies shown example, For have myogenic understood. mechanical the poorly mediates convert are that signaling responses cells signaling the biochemical satellite However, to stimulation which 2005). al., through fibers muscle et mechanisms new Wozniak form to 2005; cells, precursor (Tidball, satellite muscle as known of (MPCs), differentiation cells and proliferation activation, functions cellular the limited. various very of regulates remains understanding and that our (Wang ion mechanotransduction networks Notwithstanding, stretch-activated signaling 2006). complex and Thampatty, form mitogen- (MAPKs) the which kinases, kinases channels, include tyrosine protein proteins, mechanotransduction activated G cytoskeleton, mediate integrins, that cellular major components The cellular mechanotransduction. various triggering induce by cells responses living on applied stimuli Mechanical Introduction of soleus the and in Src impaired tyrosine are overloading, words: regeneration Key functional in and to resulting myogenesis response overloading-induced TACE, In Further, expression. with muscle. gene soleus colocalizes the myogenic mouse in Src and impaired in Tyr702 activated in residue interacts acid are resulting turn amino TACE signaling, of in TNF TACE–p38-MAPK phosphorylation increased of the the which in stretch via of resulting mechanical TACE Src, that TACE, activates observed of activates Src tyrosine We tail Particularly, MPCs. rapidly non-receptor intracellular TACE. in the of myoblasts TACE of activation of substrates and rat activation of phosphorylation mechanical primary features mediates cells Src structural or that the precursor hypothesis possesses C2C12 the TACE myogenic activating tested Because we via In unknown. Src, myogenesis, is kinase mechanotransduction. of TACE regulator through of key activation a cells mechanical (MAPK), living kinase protein in mitogen-activated TNF p38 aspects activates biological stimulation mechanical many (MPCs), affects stimulation Mechanical Summary 10.1242/jcs.125328 doi: 4349–4357 126, ß Science Cell of Journal 2013 June 30 Accepted ( correspondence for *Author 2 eateto nertv ilg n hraooy nvriyo ea elhSineCne,Hutn X700 USA 77030, USA TX 77030, Houston, TX Center, Houston, Science Medicine, Health of Texas College of Baylor University Medicine, Pharmacology, of and Department Biology Integrative of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. ehnclsiuainidcsmoeei yprovoking by myogenesis induces stimulation Mechanical +/ a 2 cnetn nye(AE lokona DM7,t ees uorn TNF autocrine release to ADAM17), as known also (TACE, enzyme -converting ie hrfr,Scmdae ehn-ciaino AEadmyogenesis. and TACE of mechano-activation mediates Src Therefore, mice. 1 ee Wen Yefei , AE DM7 r,Mcaorndcin 3 AK ygncgn xrsin uceregeneration Muscle expression, gene Myogenic MAPK, p38 Mechanotransduction, Src, ADAM17, TACE, a hc scuilt ygncatvto fthe of activation myogenic to crucial is which , [email protected] 1 ujeLiu Huijie , ´ ta. 06.Yt h mechanism the Yet, 2006). al., et s ) 1 e Zhan Mei , a cnetn enzyme -converting a 2 ige Jin Bingwen , ees n 3 ciain r niiino eiinybok tec activation stretch blocks deficiency or inhibition Src activation. p38 and release efudta TNF that found we ta. 05.I el htaemcaial tmltd Src stimulated, mechanically are mechanical Wang that 1999; non- of al., which cells rapidly, et of dephosphorylation transduction In and Sai phosphorylation variety 2004; undergoes 2005). the al., a al., et in in (Han et cells role signaling muscle biochemical skeletal a into been play has stimulation protein 1997), to Brugge, TACE and (Thomas shown membrane mediate plasma of might face which 2005). 2005), al., et (Soond al., (Dı in trafficking ERK et Thr735 2003). by dephosphorylation al., Soond phosphorylated et (Fan undergoes also stimulation is factor Ser791 growth to response whereas ERK1/2 the signaling a via is MAPK, phosphorylation Ser819 intracellular growth-factor-induced example, of For with activity. target TACE interacts regulate that 695–828) (amino substrate molecules tail integrins, residues cytoplasmic the The acid with cleavage. substrate interaction and in of recognition domains involved extracellular are The 1997). TACE al., et (Black muscle skeletal unknown. is TACE activates stimulation new mechanical TNF TACE a which autocrine of activation revealed of the via findings release expression transduced gene These myogenic are activate cues p38 2007). to myogenic which of al., through mechano-activation paradigm et signaling for limiting (Zhan rate MAPK is and stimulation pro-TNF TNF free membrane-anchored release that plasma 2002) (Black, cleaves metalloproteinase disintegrin the ADAM17), 2 r,annrcpo yoiekns hti nhrdt h inner the to anchored is that kinase tyrosine non-receptor a Src, including tissues most in expressed constitutively is TACE n iPn Li Yi-Ping and a 1 D) srpdyatvtdb mechanical by activated rapidly is kDa), (17 a cnetn nye(AE lokonas known also (TACE, enzyme -converting a 1, oee,tesgaigmcaimof mechanism signaling the However, . * a oee,temcaimthrough mechanism the However, . ´ az-Rodrı ´ uze l,2002; al., et guez a 2 D)to kDa) (26 4349 Journal of Cell Science uaietrsn hshrlto oi (K motif phosphorylation tyrosine putative r usrts–teitaellrti fTC otisapotential a (P contains of TACE motif of (SH3) features tail SH3-binding intracellular structural 3 the the – substrates homology possesses Src TACE Baltimore, Src and Interestingly, (Alexandropoulos the substrate 1996). Src the substrates and between kinase Src its of interaction domain tyrosine of phosphorylation an the cells. Src requires by muscle unknown. kinases non-skeletal largely activation remain serine/threonine of the mediate these types that of various mechanisms signaling (Jin in AKT the and However, 2005), 2005) (Nadruz al., MAPK al., et JNK (Plotkin et 2002), MAPK ERK1/2 al., 2005), et the al., (Aikawa et to stimulation, MAPK mechanical p38 leads to as Src sensitive such are of downstream that kinases activation its of is mechanical activation It activates that 1999). al., turn established et well (Sai in phosphorylation tyrosine Src through effectors activated Src; activates vdneta r eitsmcaia ciainof activation mechanical present now mediates We Src hypothesis. TACE. activating this by myogenesis that tested p38 we activates evidence putative turn study, the TNF autocrine in present releasing within through which TACE myogenesis Tyr702 and of motif, MAPK of activation phosphorylation hypothesized mechanical phosphorylation tyrosine we mediates Therefore, the Src Src. through of suggest MPCs, substrate TACE in a of that, be features might structural it These that 1997). al., et (Moss 4350 ora fCl cec 2 (19) 126 Science Cell of Journal 731 APQTPGR 738 ,wihi daett a to adjacent is which ), 696 KLDKQ Y a ESL nthe In . 705 ) Results yoiepopoyaino AEa iue fsrth and stretch, of in minutes 5 increase at an TACE observed of antibody phosphorylation We an tyrosine using tyrosine. blotting phosphorylated western against by analyzed and TACE t myoblasts TACE, from To of 1A). immunoprecipitated lysates state (Fig. was phosphorylation minutes minutes cell 60 tyrosine 30 at the around for subsiding at in determine stretched rapidly peaked before been activation seen stretch Src had of the was that and minutes, myoblasts Src 5 C2C12 mon from activated prepared blot to of western performed augmentation 1997), was Brugge, Tyr416 and (Thomas analysisusinganantibodyspecificforSrcwithphosphorylated were residue medium in Tyr416 activatio growth Src activation serum-rich Because singl periods. in 10% and to membrane subjected phosphorylation silicon myobl on TACE stretched Src between mechanically and relationship first causal we a activity stretch, is whether mechanical there assess by whether To myoblasts investigated activation 2007). 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(AWC) in Committee approved Welfare were protocols Experimental use Animal medium the to added was negative DMSO, 4-amino-5-(4- in inhibitor 10 its dissolved concentration SFK (final (Sigma-Aldrich), the or PP3 indicated, control When (PP2) incubator). chlorophenyl)-7-(t-butyl)pyrazolo[3,4-days]pyrimidine humidified a in at plated o-tece ybat eeue scnrl.Srthwsmitie o a for maintained was (37 conditions Stretch culture cell controls. normal under as time of used period designated were myoblasts non-stretched or eoeacntn 0 lblsrthwsiiitduigteFlexcell the using for initiated medium was growth stretch fresh global in 10% placed 5000 constant were a cells before point, hours this 2 At I. collagen with coated Bioflex six-well h oto.Atrtecmlto ftesrth ybat eecletdby 2007). collected al., rats et were neonatal (Chen myoblasts from described prepared previously stretch, as were the old) myoblasts days Primary of (2–4 PBS. for completion ice-cold medium the the into to scraping After added was control. concentration) final the (0.1% DMSO stretch; the during niio okal SgaAdih] ucehmgnt a hnsnctdfor 12,000 at sonicated centrifugation protease/phosphatase by then and removed was was sodium EDTA, Debris homogenate seconds. 0.1% mM 5 Muscle 2 deoxycholate, (Sigma-Aldrich)]. fluoride, sodium cocktails sodium 0.5% inhibitor mM NP-40, 2 1% sulfate, NaCl, dodecyl mM 150 Tris-HCl, tlzn etd fteptniltrsn hshrlto iei AEwith TACE in site phosphorylation tyrosine potential obtained (K the was Y702 raised of p21 phosphorylated was peptide against pY702-TACE against Antibody a antibody Iowa. utilizing Rabbit Biotechnology. of Cruz University Santa and Studies the from Laboratories. 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(day by control total analyzed with the were compared against Data normalized GAPDH. proteins or the protein of of data shown density forms sample optical activated Each summarize the p38 graphs (D). Bar and myogenin mouse. (B) of one TACE expression represents (A), as for Src well collected of as was activation (C), soleus of indicated the analysis the surgery, blot At the western 0). after (day days control of plantaris. as number and used soleus were of mice severed overloading Sham-operated was functional (C57BL/6) create soleus. mice to mouse male bilaterally overloaded adult in of activated gastrocnemius are TACE The and Src 7. Fig. TM H iu uiiainktfo Clontech. from kit purification virus ltswr ie ntepae n ujce to subjected and plates the in fixed were plates H ltso hc el eeatce a then was attached were cells which on plates Pme ,adte rnfre noBJ5183 into transformed then and I, m m. Xho P Iand , 0.05 Xba E. I Journal of Cell Science opr,M . ismn .F,Skmt,K,Fedn,R .adGoya,L J. L. Goodyear, and A. R. Fielding, K., Sakamoto, F., M. Hirshman, D., M. Boppart, li,H . oisn .J n ad,M. Zaidi, and J. L. Robinson, C., H. Blair, MCi vroddslu r uniidi h a rp.Cosscin fslu hthdbe vroddfr1 aswr tie yHE() Central (C). H&E by stained or were myogenin days express 10 for that overloaded myofibers 100 been expressio of bars: had (eMHC) percentage Scale that MHC The soleus regeneration. embryonic soleus. of of and sections of indication Cross (A) sections an graph. 5 cross as bar day of the counted on immunohistochemistry in were expression quantified by nuclei Myogenin are compared soleus gastrocnemius. were overloaded the overloading in of of eMHC ablation (B) bilateral 10 to day subjected on were littermates (WT) type lc,R . ac,C . olsy .J,Pshn .J,Sak .L,Wolfson, L., J. Slack, J., J. Peschon, J., C. Kozlosky, T., C. Rauch, A., R. Black, A. R. Black, lxnrpuo,K n atmr,D. Baltimore, and K. Alexandropoulos, iwl,J .adPvlo .M. F. Pavalko, and P. J. Bidwell, iaa . aa,T,Kdh . o,Y,Tnk,M,Tmr,M,Aaaa H., Akazawa, M., Tamura, M., Tanaka, Y., Zou, S., Kudoh, T., Nagai, R., Aikawa, and months. References 12 Arthritis after release of for Institute to PMC AR049022 in National R01 Deposited number Y.-P.L.]. by [grant Diseases supported Skin and Musculoskeletal was work This and Funding experiments designed Y.-P.L. analyzed figures. manuscript. experiments, the the conducted wrote generated B.J. and and M.Z. data H.L., Y.W., A.N., interest. contributions of Author conflict no have they that declare authors The Medicine, of College Acknowledgements (Baylor Core Pathology Center USA). were Breast TX, sections Houston, the soleus by (H&E)-stained Hematoxylin-and-eosin prepared Iowa. of University soleus. mouse in regeneration and differentiation myogenic activation, TACE induced overloading for essential is Src 8. 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