fynactivationofmtorc1stimulatestheire1 -jnk pathway… · 2015-10-02 ·...

Fyn Activation of mTORC1 Stimulates the IRE1�-JNKPathway, Leading to Cell Death*

Received for publication, August 21, 2015 Published, JBC Papers in Press, August 25, 2015, DOI 10.1074/jbc.M115.687020

Yichen Wang‡, Eijiro Yamada§, Haihong Zong¶, and Jeffrey E. Pessin‡¶1

From the Departments of ‡Molecular Pharmacology and ¶Medicine, Albert Einstein College of Medicine, Bronx, New York 10461and the §Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine,Maebashi 371-8511, Japan

Background: Skeletal muscle-specific Fyn transgenic mice show severe muscle wasting phenotype concomitant withincreased mTORC1 activity.Results: Overexpression of Fyn stimulates mTORC1 and IRE1� phosphorylation, and rapamycin treatment represses Fyn- andER stress-induced cell death.Conclusion: Fyn plays a role in ER stress-induced cell death at least partially through regulation of mTORC1.Significance: Our findings indicate that Fyn drives pro-apoptotic signaling by activating the unfolded protein response.

We previously reported that the skeletal muscle-specific over-expression of Fyn in mice resulted in a severe muscle wastingphenotype despite the activation of mTORC1 signaling. Toinvestigate the bases for the loss of muscle fiber mass, we exam-ined the relationship between Fyn activation of mTORC1, JNK,and endoplasmic reticulum stress. Overexpression of Fyn inskeletal muscle in vivo and in HEK293T cells in culture resultedin the activation of IRE1� and JNK, leading to increased celldeath. Fyn synergized with the general endoplasmic reticulumstress inducer thapsigargin, resulting in the activation of IRE1�and further accelerated cell death. Moreover, inhibition ofmTORC1 with rapamycin suppressed IRE1� activation and JNKphosphorylation, resulting in protecting cells against Fyn- andthapsigargin-induced cell death. Moreover, rapamycin treat-ment in vivo reduced the skeletal muscle IRE1� activation in theFyn-overexpressing transgenic mice. Together, these data dem-onstrate the presence of a Fyn-induced endoplasmic reticulumstress that occurred, at least in part, through the activation ofmTORC1, as well as subsequent activation of the IRE1�-JNKpathway driving cell death.

The endoplasmic reticulum (ER)2 is responsible for the fold-ing, maturation, and trafficking of most secretory and mem-brane proteins, as well as for autophagosome biogenesis, gluco-neogenesis, and lipid synthesis (1). ER stress happens when the

nascent protein loading exceeds the ER folding capacity undercircumstances such as virus infection, gene mutation, calciumflux perturbation, or glucose deprivation, and it has been linkedwith diseases such as cancer, neurodegeneration, inflamma-tion, metabolism, aging, and muscle dysfunction (2, 3). ERstress activates the unfolded protein response (UPR) to sup-press general protein synthesis and increase the ER foldingcapacity and misfolded protein degradation, thereby restoringthe cell back to homeostasis. However, if the stress response isprolonged or beyond the adaptive range, UPR may also lead tocell death (1).

The UPR is mediated by three pathways, RNA-dependentprotein kinase-like ER kinase (PERK), activating transcriptionfactor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) (4, 5).PERK is an ER-resident type I transmembrane protein kinase.PERK phosphorylation at Ser-51 of eukaryotic translation ini-tiation factor 2� (eIF2�) in general inhibits cap-dependent pro-tein translation with the notable exception of activating tran-scription factor 4 (ATF4). As a transcription factor, ATF4expression is activated by phosphorylation of eIF2�, which inturn promotes the transcription of CCAAT/enhancer-bindingprotein homologous protein (CHOP), which is important in ERstress-induced apoptosis by regulating calcium signaling andcytochrome c release from mitochondria (6). Initially located atthe ER as a type II transmembrane protein, ATF6 is transferredto the Golgi, where it goes through cleavage by site-1 proteaseand site-2 protease. The cleaved N-terminal ATF6 fragmententers the nucleus and increases the transcription of adaptivechaperons, such as Bip. IRE1 is a type I transmembrane proteinwith both endoribonuclease and kinase activity. IRE1� is ubiq-uitously expressed, whereas the other isoform, IRE1�, is foundonly in the epithelial cells of gastrointestinal track (7) and air-way (8). The endoribonuclease activity of IRE1� cleaves a26-nucleotide intron from X-box-binding protein 1 (XBP1) togenerate spliced XBP1 (XBP1s), a potent transcription factorthat participates in pro-survival response but that declines dur-ing prolonged progression of ER stress (9). IRE1� also forms acomplex with the adaptor protein TNFR-associated factor2 (TRAF2) to activate apoptosis signal-regulating kinase 1

* This work was supported by National Institutes of Health Grants DK033823,DK098439, and DK020541 (to J. E. P.). The authors declare that they haveno conflicts of interest with the contents of this article.

1 To whom correspondence may be addressed: Dept. of Medicine and Molec-ular Pharmacology, Albert Einstein College of Medicine, 1300 Morris ParkAve., Bronx, NY 10461. Tel.: 718-678-1029; Fax: 718-678-1020; E-mail:[email protected].

2 The abbreviations used are: ER, endoplasmic reticulum; UPR, unfolded pro-tein response; PERK, RNA-dependent protein kinase-like ER kinase; ATF,activating transcription factor; CHOP, CCAAT/enhancer-binding proteinhomologous protein; IRE1, inositol-requiring enzyme 1; XBP1, X-box-bind-ing protein 1; mTOR, mechanistic target of rapamycin; mTORC1, mTORcomplex 1; mTORC2, mTOR complex 2; S6K, S6 ribosomal protein kinase;AMPK, AMP-activated protein kinase; TG, thapsigargin; TNFR, TNF recep-tor; KD, kinase-defective; SH, Src homology; PI, propidium iodide.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 41, pp. 24772–24783, October 9, 2015

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

24772 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 41 • OCTOBER 9, 2015

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(ASK1) and JNK (10). The activation of JNK is considered to bepro-apoptotic as it phosphorylates different members of theBcl-2 family and shifts the balance toward cell death (6).

Mechanistic target of rapamycin (mTOR) (previouslytermed mammalian target of rapamycin) is a master manipula-tor of cell growth and metabolism. This atypical serine/threo-nine protein kinase is the core of two distinct multi-proteincomplexes, mTOR complex 1 (mTORC1) and mTOR complex2 (mTORC2), which share common core subunits that includemTOR, mammalian lethal with sec-13 (mLST8), DEP domaincontaining mTOR-interacting protein (DEPTOR), and theTti1-Tel2 complex (11). In addition, the mTORC1 complexalso contains the regulatory-associated protein of mammaliantarget of rapamycin (Raptor) and proline-rich Akt substrate 40kDa (PRAS40), whereas the mTORC2 complex contains therapamycin-insensitive companion of mTOR (Rictor), mamma-lian stress-activated map kinase-interacting protein 1 (mSin1),and protein observed with Rictor 1 and 2 (protor1/2) (11).mTORC1 enhances protein synthesis through direct phos-phorylation of the eukaryotic translation initiation factor4E-binding protein 1 (4EBP1) and p70 S6 kinase (S6K), andribosomal protein S6 is a downstream target of S6K (12).

Fyn is a ubiquitously expressed non-receptor tyrosine kinaseof the Src family (13). Two intramolecular interactions, SH3-linker and SH2-phosphorylated tyrosine (SH2-pY) tail, main-tain Fyn in a closed inactive conformation (14). Although Fynfunctions in B cell expansion and maturation (15, 16) andtumor progression and metastasis (17), it also regulates fattyacid oxidation through direct phosphorylation of liver kinase B1 (LKB1) and inactivation of the AMP-activated protein kinase(AMPK) (18 –20). In turn, inhibited AMPK dampens its func-tion in phosphorylating Raptor and tuberous sclerosis proteins1 and 2 complex (TSC1/2 complex, the upstream RhebGTPase-activating protein) to suppress mTORC1 activation(21).

Thapsigargin (TG) induces ER stress through ER calciumdepletion by ER calcium ATPase blockage (22). It was reportedthat mTORC1 was activated under ER stress conditions andthat its activation led to cell death via IRE1�-JNK pathway (23).Consistently, TSC1- or TSC2-deficient cells showed increasedphosphorylation of S6, IRE1�, and JNK and were sensitive to ERstress-induced apoptosis, which was inhibited by rapamycintreatment (24).

We previously reported that Fyn deficiency activates fattyacid oxidation with improved glucose homeostasis in wholebody Fyn knock-out mice (25) and that Fyn overexpressionactivates mTORC1 through inhibition of the LKB1-AMPKpathway (18, 26). Moreover, skeletal muscle-specific Fyn trans-genic mice (SKM-Fyn) displayed a marked phenotype of musclewasting (26). Based upon these findings, we speculated that Fynfunctions as an inducer of ER stress through the indirect acti-vation of mTORC1, and the consequent activation of IRE1�pathway and cell death can at least partially explain the musclewasting in SKM-Fyn mice. In the present study, we demon-strate that both in transfected cells in culture and in transgenicmice, Fyn overexpression activates mTORC1 with the concom-itant activation of IRE1�-JNK pathway, which potentiates theER stress-induced cell death.

Experimental Procedures

Antibodies and Reagents—The phospho-IRE1� antibody waspurchased from Novus Biologicals (Littleton, CO). Antibodiesfor detecting GAPDH and V5 were purchased from MarineBiological Laboratory (Woods Hole, MA), and all the otherantibodies were purchased from Cell Signaling (Boston, MA).Hoechst was purchased from Life Technologies. Propidiumiodide, thapsigargin, and tunicamycin were obtained from Sig-ma-Aldrich. Rapamycin for in vitro experiments was purchasedfrom Sigma-Aldrich, and rapamycin for in vivo injection waspurchased from LC Laboratories (Woburn, MA). JNK inhibitorwas purchased from Calbiochem.

Fyn Transgenic Mice—The skeletal muscle-specific FynBtransgenic and littermate control mice were generated asdescribed previously and maintained on a C57BL6/129svj back-ground (26). The mice were fed ad libitum with chow diet con-taining 20% protein and 9% fat (PicoLab Rodent Diet 20, catalognumber 5058). All animal studies were approved by and per-formed following the guidelines from the Institutional AnimalCare and Use Committee (IACUC) of Albert Einstein Collegeof Medicine.

Cell Culture—HEK293T cells (ATCC, Manassas, VA) werecultured in Dulbecco’s modified Eagle’s medium (Invitrogen)with 10% fetal bovine serum (Atlanta Biologicals, FloweryBranch, GA) and 1% penicillin-streptomycin (Life Technolo-gies). Cells were incubated at 37 °C in a moisturized incubatorwith 5% CO2. Cell transfection was performed using theFuGENE HD transfection reagent according to the manufac-turer’s instructions (Promega, Fitchburg, WI). Plasmids ofpcDNA, pcDNA-Fyn-WT-V5, and pcDNA-Fyn-KD-V5 wereconstructed before (26).

Hoechst, Propidium Iodide, and TUNEL Analysis—Hoechstreagent and propidium iodide at 4 and 1 �g/ml were applied tothe cells grown in 6-well plates followed by incubation at 37 °Cfor 5–10 min. The incorporation of label was determined byfluorescent light microscopy. TUNEL assay was applied to thecells grown on coverslips after 4% paraformaldehyde fixation,followed by using the ApopTag� fluorescein in situ apoptosisdetection kit as described by the manufacturer’s instructions(Millipore, Billerica, MA).

Microscopic Analysis—The live cells were directly observedin 6-well plates under transmitted light microscopy using aZeiss Axiovert 40c microscope with a 10� A plan 0.25 objec-tive, equipped with a Canon PowerShot A640 digital camera.Fluorescent images for Hoechst and propidium iodide andcorresponding translight images were obtained from livecells using an Olympus 1X71 inverted microscope with aLUCPlanFLN 10� 0.30 or 20� 0.45 objective and exportedby Olympus DP2-BSW application software. Fluorescentimages of TUNEL staining were obtained using a Leica SP5AOBS confocal microscope with a 63� 1.4 oil objective andexported by the Leica LAS-AF software. All images werecaptured at room temperature.

Immunoblotting—Cell and tissue extracts were prepared inlysis buffer as described by Cao et al. (27) containing proteaseinhibitor cocktail set V (Calbiochem) and phosphatase inhibi-tor cocktail 2 (Sigma-Aldrich). Protein concentration was mea-

Fyn Activation of the mTORC1-IRE1�-JNK Pathway

OCTOBER 9, 2015 • VOLUME 290 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 24773


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sured using the BCA protein assay kit (Thermo Scientific).Total lysates were separated in 8 or 15% SDS-PAGE overnightor semidry transferred to PVDF membranes at 4 °C and soakedin blocking buffer (GenDEPOT, Barker, TX) for 2 h at roomtemperature. The incubation of primary antibodies proceededat 4 °C overnight, and the incubation of secondary antibodies(Thermo Scientific and LI-COR Biosciences, Lincoln, NE) pro-ceeded at room temperature for 30 min before developmentusing ECL or the Odyssey infrared imaging system (9140-01Odyssey� CLx infrared imaging system, LI-COR Biosciences).

Semi-quantitative and Real-time Quantitative PCR—TheRNeasy mini kit (Qiagen, Valencia, CA) was used to isolate totalRNA from cells or tissues. cDNA was generated by reverse tran-scription using a SuperScript VILO cDNA synthesis kit (Invit-rogen) and served as a template for semi-quantitative and quan-titative PCR. Semi-quantitative PCR for detecting XBP1splicing was performed by using the GoTaq Green master mix(Promega) on a T1000TM thermal cycler (Bio-Rad). Primersequences for XBP1 and PCR condition were described previ-ously (9), and GAPDH was amplified as internal controltogether with the following primers: forward, 5�-ACC ACAGTC CAT GCC ATC AC-3�, and reverse, 5�-TCC ACC ACCCTG TTG CTG TA-3�. PCR products were resolved by elec-trophoresis on a 2.5% agarose (Bio-Rad)/Tris-acetate-EDTAgel. Real-time quantitative PCR reactions were run on a7900HT Fast Real-Time PCR system with a 384-well blockmodule (Life Technologies). Gene expression of Bip, Chop,Fyn, and GAPDH was amplified by using TaqMan gene expres-sion assays with the ��Ct method for quantification (AppliedBiosystems, Branchburg, NJ). Spliced XBP1 was amplified bythe Integrated DNA Technologies PrimeTime quantitativePCR assay (Integrated DNA Technologies, Coralville, IA) asdescribed previously (28, 29). GAPDH was amplified in eachexperiment and served as the endogenous internal control.

Generation of Stable Knockdown Cell Lines—The stable cellline with endogenous IRE1� knockdown was achieved by len-tivirus infection using a pLKO.1 vector containing IRE1�-tar-geting shRNA (TRCN0000000530). Control cells were infectedby lentivirus containing the empty vector (pLKO.1). Virus gen-eration and cell infection were performed following the proto-col from Addgene. Puromycin (2.5 �g/ml) was applied after24 h of infection for cell selection of positive infection.

Quantification and Statistical Analysis—The numbers of thefluorescent puncta from microscopy analysis were counted byusing the ImageJ software (National Institutes of Health,Bethesda, MD). All the results present were representativesfrom at least three repeats, and quantified data were present asmean � S.E. Significant data were defined as p � 0.05 usinganalysis of variance followed by the Tukey’s multiple compari-son or Student’s t test.

Results

Fyn Overexpression in Skeletal Muscle Increases ER Stress—Previously, we observed that transgenic mice overexpressingFyn in skeletal muscle resulted in a marked increase ofmTORC1 activation (26), which is important in regulation ofER stress-induced cell death (23, 24). To investigate the corre-lation between Fyn- and ER stress-induced cell death, in whichthe latter could be responsible for the muscle wasting pheno-type of SKM-Fyn mice, we examined the effect of Fyn on theindicative markers of three UPR pathways. The Western blotanalyses of SKM-Fyn mice demonstrated a marked increasein IRE1� phosphorylation (Fig. 1A). The increased IRE1�phosphorylation was associated with the increase in XBP1splicing and JNK phosphorylation (Fig. 1, A and C). In par-allel, the downstream targets of ATF6 and PERK stimulation(Bip and Chop mRNA, respectively) were also elevated, indi-cating that all three arms of the UPR were activated (Fig. 1C).

FIGURE 1. Skeletal muscle-specific Fyn overexpression induces mTORC1 and IRE1� activation. Wild type (WT) and SKM-Fyn mice (Tg) at 3 weeks of agewere sacrificed after an overnight fast. A and B, gastrocnemius muscles were isolated, and cell extracts were prepared for Western blotting against the proteinsindicated. These are representative immunoblots independently performed three times. p indicates phosphorylated form. C, the total RNA was extracted fromisolated gastrocnemius muscles. The mRNA levels of XBP1s, Bip, and Chop were determined by quantitative PCR. The data are presented as mean � S.E. fromfour independent experiments. *, p � 0.05; **, p � 0.01.




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Thr-389 is the mTORC1-specific site of S6K phosphoryla-tion, which is the major event promoting S6K activation (30).Consistent with our previous results, Fyn overexpressionalso led to the mTORC1 activation (26), shown by theincreased phosphorylation of S6K at Thr-389 and its down-stream S6 phosphorylation (Fig. 1A). 4EBP1 is another directmTORC1 substrate that is phosphorylated on multiple sites,represented by three bands (�, �, �) in SDS-polyacrylamidegels (31, 32). In accordance with increased S6K phosphory-lation, SKM-Fyn transgenic mice also demonstrated in-creased � and � bands with decreased � band indicative ofmTORC1 activation (Fig. 1B).

Fyn Overexpression Activates mTORC1 and IRE1� inHEK293T Cells in Culture—To examine the role of Fyn inmediating ER stress in a more experimentally tractable system,we transfected wild type Fyn (Fyn WT) and a kinase-defectiveFyn mutant (Fyn KD) in HEK293T cells. Expression of Fyn WTincreased IRE1� and JNK phosphorylation as well as that of themTORC1 substrate S6K (Fig. 2A). In contrast, expression ofFyn KD had no significant effect (Fig. 2A). Neither expression ofFyn WT nor expression of Fyn KD had any statistically signifi-cant effect on Bip or Chop mRNA levels (Fig. 2, B and C). Thesedata indicate that the effect of Fyn overexpression on IRE1�phosphorylation in mice is recapitulated in acute Fyn-trans-fected HEK293T cells. However, the lack of effect on Bip or

Chop probably reflects the additional presence of ER stressinducers in vivo that are not present in tissue culture. Toaddress this possibility, HEK293T cells were transfected withand without the Fyn and subsequently treated with TG, a wellestablished ER stress inducer (33). As expected, TG inducedboth Bip and Chop mRNA that were not further increased bythe expression of Fyn WT or Fyn KD (Fig. 3, A and B). AlthoughTG treatment also increased XBP1 splicing, this response wasfurther enhanced by the expression of Fyn WT (Fig. 3, C and D).These data indicate that Fyn regulates the IRE1� signalingpathway of UPR.

Fyn Overexpression Induces Cell Death in HEK293T Cellsin Culture through IRE1�-JNK Pathway—As visually appar-ent, acute expression of Fyn resulted in significant morpho-logical changes that include cell shrinkage, increased round-ing, and reduced adherence to the substratum (Fig. 4A). Incontrast, expression of Fyn KD had no significant morpho-logical features when compared with empty vector control-transfected cells. To assess whether the Fyn WT-inducedmorphology was associated with cell death, the cells weretreated with Hoechst to visualize condensed nuclei and pro-pidium iodide (PI) to assess cell permeability. Hoechst stain-ing demonstrated a large increase in condensed nuclei cells,and PI demonstrated a large increase in permeable cells fol-lowing acute Fyn WT transfection. Quantification of the PI

FIGURE 2. Fyn activates mTORC1, IRE1�, and JNK in HEK293T cells. HEK293T cells were transfected with the empty vector (pcDNA), Fyn wild type (FynWT), or Fyn kinase-defective (Fyn KD). A, forty-eight hours after transfection, cell extracts were prepared and subjected to Western blot for the indicatedproteins. These are representative immunoblots independently performed three times. p indicates phosphorylated form. B, RNA was prepared, and thelevels of Bip and Chop mRNA were determined by quantitative RT-PCR. These data are presented as mean � S.E. from three independent experiments.




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staining is shown in Fig. 4B. The Fyn WT- but not Fyn KD-induced cell death was further confirmed by TUNEL staining(Fig. 5, A and B).

To assess the IRE1� dependence of Fyn-induced cell death,we generated a stable cell line with IRE1� deficiency by infec-tion with a lentivirus shRNA and a control cell line infected

FIGURE 3. TG increases Fyn induction of XBP1s but not Bip or Chop mRNA expression in HEK293T cells. HEK293T cells were transfected with the emptyvector (pcDNA), Fyn wild type (Fyn WT), or Fyn kinase-defective (Fyn KD). Forty-eight hours later, the cells were treated with 1 �M TG for 4 h. A–C, the levels ofBip (A), Chop (B), and spliced XBP1 (XBP1s) (C) mRNA were determined by quantitative RT-PCR. D, the mRNA levels of unspliced XBP1 (XBP1u) and spliced XBP1(XBP1s) were analyzed by semi-quantitative PCR. These data are presented as mean � S.E. from three independent experiments. Non-identical letters (a, b, c,and d) indicate results that are statistically different from each other at p � 0.05.

FIGURE 4. Overexpression of active Fyn kinase in HEK293T cells increases Hoechst and propidium iodide staining. HEK293T cells were transfected withthe empty vector (pcDNA), Fyn wild type (Fyn WT), or Fyn kinase-defective (Fyn KD). A, forty-eight hours later, the cells were subjected to Hoechst and PI staining.These are representative images from experiments independently performed three times. B, the numbers of positive PI-stained cells were quantified from 10images. These data are presented as mean � S.E. Non-identical letters (a and b) indicate results that are statistically different from each other at p � 0.01.




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with the empty lentiviral vector (pLKO.1). PI staining demon-strated a lower degree of Fyn-induced cell death in the IRE1�knockdown cells (Fig. 6, A and B).

JNK phosphorylation is a downstream consequence of IRE1�activation leading to cell death through phosphorylation ofBcl-2 family members (6). Consistent with the IRE1� depen-dence of JNK phosphorylation, knockdown of IRE�1 reducedthe extent of JNK phosphorylation that occurs in Fyn-overex-pressing cells (Fig. 6C). Treatment of Fyn-transfected cells witha JNK inhibitor resulted in fewer condensed nuclei and PI-pos-itive stained cells (Fig. 7, A and B). Together these data indicatethat the Fyn-induced cell death occurs at least partially throughactivation of the IRE1�-JNK pathway.

Thapsigargin Potentiates Cell Death Induced by FynOverexpression—To examine the effects of general ER stresswith Fyn, we next examined cell death by TG in cells overex-pressing Fyn. As observed previously, following transfectionwith Fyn WT but not Fyn KD, there were obvious visual mor-phological changes indicative of cell death initiation (Fig. 8).Treatment of control cells with TG also induced some morpho-logical changes that were not as visually apparent when com-pared with that observed in the Fyn WT-transfected cells. Inany case, the combination of TG treatment in cells overexpress-ing Fyn WT displayed a large increase in cell detachment withthe remaining cells highly rounded, indicative of cell death.

As observed previously, expression of Fyn WT increasedIRE1� and JNK phosphorylation along with increased phos-phorylation of the mTORC1 downstream targets, S6K and S6

(Fig. 9A). Although TG treatment also activated these path-ways, it was not as strong as Fyn WT and was further potenti-ated in the cells overexpressing Fyn WT treated with TG. Sim-ilarly, TG induced XBP1 splicing that was enhanced in cellsoverexpressing Fyn WT (Fig. 9B). Previous studies havereported that inhibition of mTORC1 with rapamycin can sup-press the ER stress response (23, 24). Consistent with thesestudies, we also observed that rapamycin suppressed the phos-phorylation of IRE1� and JNK as well as the expected mTORC1target substrates (Fig. 9A). The inhibition of IRE1� activationwas also observed by the inhibition of XBP1 splicing (Fig. 9B).However, rapamycin did not significantly inhibit the inductionof Bip and Chop mRNA, further supporting a specific effect ofFyn on the IRE1� pathway through the activation of mTORC1(Fig. 9C).

As shown in Fig. 8, thapsigargin treatment of Fyn WT-trans-fected cells resulted in a substantial detachment of cells char-acteristic of cell death. However, rapamycin treatment com-pletely protected against the Fyn WT-induced loss of cells (Fig.10). Moreover, rapamycin treatment of the SKM-Fyn trans-genic mice in vivo not only inhibited the Fyn-induced activationof mTORC1 (S6 phosphorylation) but also suppressed IRE1�and JNK phosphorylation (Fig. 11A) and resulted in a concom-itant decrease of XBP1s, Bip, and Chop mRNA levels (Fig. 11,B–D). These data are consistent with a Fyn-induced activationof mTORC1 that drives ER stress-induced cell death, at leastpartially through IRE1�.

FIGURE 5. Overexpression of active Fyn kinase in HEK293T cells increases TUNEL staining. HEK293T cells were transfected with the empty vector (pcDNA),Fyn wild type (Fyn WT), or Fyn kinase-defective (Fyn KD). A, forty-eight hours later, the cells were subjected to TUNEL staining. These are representative imagesfrom experiments independently performed three times. DAPI was pseudocolored as blue, whereas terminal deoxynucleotidyltransferase (TdT) was pseudo-colored as green. B, the percentage of terminal deoxynucleotidyltransferase (TdT)-positive cells was quantified from 10 images. These data are presented asmean � S.E. Non-identical letters (a and b) indicate results that are statistically different from each other at p � 0.01.




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Discussion

Skeletal muscle is the largest organ of mammals. It under-takes indispensable functions in locomotion, posture,breathing, and whole body metabolism (34). Calcium signal-ing profoundly participates in skeletal muscle physiology ofcontraction and pathology of muscle dystrophy (35, 36),which puts ER, the reservoir of calcium, in a more vital posi-tion in skeletal muscle than in other tissues. For example,loss of skeletal muscle mass with decreased strength is amajor contributor to frailty that occurs during aging (37),which is correlated with increased ER stress (38). In addition,other muscle wasting pathologies also occur during states ofdenervation and immobilization (disuse-related atrophy)and in states of cancer cachexia, sepsis, and diabetes (nutri-ent-related atrophy). Several pathways have been identifiedas stimulating skeletal muscle wasting in different patho-physiologic conditions including activation of the atrogin E3ligase responsible for proteasome-mediated degradation,STAT3 activation during cancer cachexia, and inhibition ofmacroautophagy (26, 39 – 41). Previously, we observed thatskeletal muscle-specific overexpression of Fyn also resulted

in severe muscle wasting associated with mTORC1 activa-tion and inhibition of AMPK activation (26). Although Fynexpression also suppressed macroautophagy, the causalityrelationship between mTORC1 activation and skeletal mus-cle wasting was not determined.

In this regard, it was shown that the loss of the tuberoussclerosis complex, an upstream inhibitor of mTORC1, inducedthe ER unfolded response to activate apoptosis (24). AsmTORC1 integrates macroautophagy (42) and ER stress sig-nals, we speculated that Fyn activation of mTORC1 would con-tribute to ER stress induction mediating UPR and skeletal mus-cle wasting. To test this hypothesis, we examined the ER stressresponses in skeletal muscle overexpressing Fyn and observedactivation of mTORC1 along with all three branches of theUPR. Moreover, treatment of the SKM-Fyn mice with the spe-cific mTORC1 inhibitor, rapamycin, suppressed UPR. Al-though Fyn expression in HEK293T cells also induced UPR, inthis case, the IRE1� branch was the only one activated. Severalgroups have observed a role for mTORC1 in activating UPR,and whether this occurs for all three branches or selectively forthe IRE1�-JNK pathway is still under debate (23, 43). Neverthe-

FIGURE 6. Knockdown of endogenous IRE1� in HEK293T cells suppresses the Fyn overexpression-induced cell death. Cells of IRE1� knockdown (IRE1�KD) and control cells (pLKO.1) were transfected by empty vector (pcDNA) or Fyn wild type (Fyn WT). A, forty-eight hours later, the cells were subjected to Hoechstand PI staining. These are representative images from experiments independently performed three times. B, the numbers of positive PI-stained cells werequantified from 10 images. These data are presented as mean � S.E. Non-identical letters (a, b, and c) indicate results that are statistically different from eachother at p � 0.05. C, cell extracts were prepared and subjected to Western blot for detection of the indicated proteins. p indicates phosphorylated form. Thesedata are representatives of three independent experiments.




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less, mTORC1 activation of UPR has been shown to inducecellular apoptosis (23). In this regard, we also observed a Fyn-dependent induction of cellular apoptosis that was suppressed

by rapamycin treatment. Moreover, TG treatment increasedUPR and potentiated the Fyn induction of apoptosis that wasprevented by rapamycin.

FIGURE 7. Inhibition of JNK represses the Fyn overexpression-induced cell death in HEK293T cells. Cells were transfected by empty vector (pcDNA) andFyn wild type (Fyn WT). A, thirty-two hours after transfection, the cells were treated with or without JNK inhibitor (JNKi, 25 �M) for 16 h before being subjectedto Hoechst and PI staining. These are representative images from experiments independently performed three times. B, the numbers of positive PI-stained cellswere quantified from 10 images. The data are presented as mean � S.E. Non-identical letters (a, b, and c) indicate results that are statistically different from eachother at p � 0.05.

FIGURE 8. Fyn potentiates TG-induced cell death in HEK293T cells. HEK293T cells were transfected with the empty vector (pcDNA), Fyn wild type (Fyn WT),or Fyn kinase-defective (Fyn KD). Forty-eight hours later, the cells were treated with 1 �M TG for 32 h. Representative transmitted light microscopy images fromthree independent experiments are presented.




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It is well accepted that the IRE1� plays an important role incell fate determination under ER stress, and IRE1� phosphory-lation is required for its activity (44), including XBP1 splicingand JNK phosphorylation (23, 45). Currently, we do not knowthe basis for the differential effect of Fyn expression on thethree UPR branches in skeletal muscle versus culturedHEK293T cells. One difference between these two systems isthe timeframe of Fyn expression. In vivo, Fyn was expressed atthe beginning of skeletogenesis and remained expressed duringembryonic development and into adulthood. In contrast, theHEK293T cells were transiently transfected with Fyn that wasexpressed for only a few days. Thus, compensational crosstalkbetween UPR branches may be activated only under chronic orprolonged stress conditions. It is also possible that the effect ofFyn activation of mTORC1, as well as the subsequent UPR acti-vation, occurs in a cell context-dependent manner.

In addition, rapamycin as an effective inhibitor of mTORC1was only partially effective in inhibiting JNK activation inHEK293T cells. This suggests the presence of an mTORC1-independent pathway responsible for JNK activation. In this

regard, the small GTP-binding protein RhoA has also beenreported to promote apoptosis via JNK signaling (46).

In any case, JNK is a stress-activated kinase and a down-stream target of mTORC1 activation that is associated withinsulin resistance (47) and aging (34). JNK activation is also apotent activator of cellular apoptosis (48), and our data demon-strate that Fyn stimulates JNK activation site phosphorylationin vitro and in vivo. Moreover, treatment with a JNK inhibitorpartially repressed Fyn-induced cell death in vitro. These find-ings provide strong evidence that Fyn activates the IRE1�-JNKsignaling pathway and that it is the JNK activation that is theproximal event responsible for inducing cell death. Addition-ally, we have also observed that during aging, the skeletal mus-cle levels of Fyn protein are increased with increased mTORC1activation,3 consistent with disrupted ER homeostasis in agedanimals (38, 49), suggesting the involvement of a Fyn-mTORC1-IRE1� pathway-induced cell death in sarcopenia(age-related muscle atrophy).

3 Y. Wang, E. Yamada, H. Zong, and J. E. Pessin, unpublished results.

FIGURE 9. Fyn potentiates TG-induced mTORC1-IRE1� activation in HEK293T cells. HEK293T cells were transfected with either the empty vector (pcDNA)or Fyn wild type (Fyn). Forty-eight hours later, the cells were treated with 1 �M TG with or without 100 nM rapamycin (Rapa) for the indicated times. A, cellextracts were prepared and subjected to Western blotting against the proteins indicated. These are representative immunoblots independently performedthree times. p indicates phosphorylated form. B, RNA was isolated, and mRNA levels of unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1s) were analyzed bysemi-quantitative RT-PCR. These are representative PCR reactions independently performed three times. C, the levels of Bip and Chop mRNA in cells treatedwith TG with or without rapamycin for 4 h were determined by quantitative RT-PCR. The data are presented as mean � S.E. from three independentexperiments.




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Taken together, these data indicate a novel role for Fyn as anactivator of ER stress mediated through IRE1� and JNK activation.As unresolved ER stress and JNK activation are established activators

of cellular apoptosis, this can account for the skeletal muscle wastinginduced by Fyn expression. Future studies are now needed to molec-ularly determine the basis for the loss of muscle fiber protein.

FIGURE 10. Rapamycin protects against Fyn and thapsigargin-induced cell death in HEK293T cells. HEK293T cells were transfected with either the emptyvector (pcDNA) or Fyn wild type (Fyn). Forty-eight hours later, the cells were treated with 1 �M TG with or without 100 nM rapamycin for 32 h. Representativetransmitted light microscopy images are presented from three independent experiments.

FIGURE 11. Rapamycin suppresses mTORC1, IRE1�, and JNK activation in SKM-Fyn mice. Three-week-old wild type (WT) and SKM-Fyn transgenic (Tg) micewere intraperitoneally injected with vehicle or rapamycin (2 mg/kg) once a day for 4 days. The mice were then fasted overnight, and gastrocnemius muscleswere isolated. A, extracts were prepared and subjected to Western blotting against the proteins indicated. These are representative immunoblots indepen-dently performed four times. p indicates phosphorylated form. B–D, extracts were prepared, and the levels of spliced XBP1 (XBP1s), Bip, and Chop mRNA weredetermined by quantitative RT-PCR. The data are presented as mean � S.E. from four independent experiments. Non-identical letters (a and b) indicate resultsthat are statistically different from each other at p � 0.05.




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Author Contributions—Y. W. and J. E. P. designed the study andwrote the manuscript. Y. W. performed and analyzed the experi-ments. E. Y. and H. Z. provided technical assistance for the prepara-tion of Figures 1, 3, and 11. E. Y. and H. Z. revised the manuscriptcritically.

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Yichen Wang, Eijiro Yamada, Haihong Zong and Jeffrey E. PessinDeath

-JNK Pathway, Leading to CellαFyn Activation of mTORC1 Stimulates the IRE1

doi: 10.1074/jbc.M115.687020 originally published online August 25, 20152015, 290:24772-24783.J. Biol. Chem.

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