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,,2
* Musculoskeletal Disease Center,
Laboratory for Skeletal Muscle Physiology and Neurobiology, Jerry L. Pettis Memorial Veterans Administration Medical Center, Loma Linda, California, USA; and
Loma Linda University School of Medicine, Loma Linda, California, USA
2Correspondence: Laboratory for Skeletal Muscle Physiology and Neurobiology, Jerry L. Pettis Memorial Veterans Administration Medical Center, 11201 Benton St. (151), Loma Linda, CA 92357, USA. E-mail: ashok.kumar2{at}va.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin-1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphorylation of Akt kinase and its downstream targets GSK-3ß, FOXO1, mTOR, and p70S6K. Furthermore, TWEAK increased the activation of NF-
B transcription factor in myotubes. Adenoviral-mediated overexpression of IB
N (a degradation-resistant mutant of NF-B inhibitory protein IB) in myotubes blocked the TWEAK-induced degradation of MyHCf. Chronic administration of TWEAK in mice resulted in reduced body and skeletal muscle weight with an associated increase in the activity of ubiquitin-proteasome system and NF-B. Finally, muscle-specific transgenic overexpression of TWEAK decreased the body and skeletal muscle weight in mice. Collectively, our data suggest that TWEAK induces skeletal muscle atrophy through inhibition of the PI3K/Akt signaling pathway and activation of the ubiquitin-proteasome and NF-B systems.—Dogra, C., Changotra, H., Wedhas, N., Qin, X., Wergedal, J. E., Kumar, A. TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
Key Words: skeletal muscle atrophy • atrogin-1 • MuRF1 • NF-B • Akt
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Muscle atrophy occurs as a result of an imbalance between anabolic and catabolic processes (1)
. Accumulating evidence suggests that a common set of biochemical changes augments the cell capacity for protein breakdown in different types of atrophying muscle (2
, 3)
. Studies of experimental animals and patients have consistently demonstrated that the ubiquitin-proteasome system is the primary regulator of protein breakdown in various types of atrophies (4
, 5)
. Recently it was shown that increased expression of one or more of three muscle-specific, ubiquitin-conjugating enzymes (E3 ligases)—atrogin-1 (also known as MAFbx), muscle RING finger protein 1 (MuRF1), and E3II—are responsible for degrading the bulk of muscle proteins in unrelated conditions (3
, 5
6
7)
. Furthermore, skeletal muscle mass is controlled by coordinated action of key regulatory pathways, including the phosphoinositide-3 kinase (PI3K)/Akt and NF-kappa B (NF-B) (1
, 8
, 9)
.
Several lines of evidence suggest that proinflammatory cytokines such as tumor necrosis factor (TNF)-, interleukin (IL) -1ß, IL-6, and IFN-
play a key role in the development of muscular abnormalities resulting in a loss of muscle mass and function (4
, 10)
. It has been reported that 1) inflammatory cytokines such as TNF- and IL-1ß inhibit skeletal muscle differentiation in vitro (11
12
13)
; 2) TNF- and other inflammatory cytokines are elevated under conditions that lead to muscle atrophy, such as cachexia induced by bacteria, HIV, chronic heart failure, and cancer (10
, 14
15
16)
; and 3) proinflammatory cytokines induce the degradation of specific muscle proteins in differentiated myotubes (17
18
19
20)
. Although inhibiting the activity of some of these cytokines has been found to ameliorate muscle atrophy in some conditions, there was no effect of neutralization of one or more of these cytokines in many diseases where even their serum levels were higher (16
, 21
, 22)
. This suggests that the classical proinflammatory cytokines cannot be the sole mediators of muscle loss in all conditions and that other cytokines may play an additional role. Identification of such cytokines/molecules and understanding their mode of action leading to loss of skeletal muscle mass is important for the development of new therapeutics.
TNF-related weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that acts on responsive cells via binding to fibroblast growth factor-inducible14 (Fn14), a small cell surface receptor (23
24
25)
. TWEAK is primarily expressed as a 249 amino acid (aa) type II transmembrane protein that is proteolytically processed into a soluble biologically active 156 aa cytokine (23)
. Soluble TWEAK protein has been shown to induce a variety of biological responses, including cellular growth and proliferation (26
, 27)
, angiogenesis (28)
, osteoclastogenesis (29)
, and stimulation of apoptosis under some experimental conditions (23
, 30)
. Recently, Maecker et al. (31)
demonstrated that, in contrast to TNF-, TWEAK attenuates the transition from innate to adaptive immunity. TWEAK also activates both the classical and alternative NF-B signaling pathways and induces the expression of NF-B-regulated proinflammatory cytokines and cell adhesion molecules (32
33
34
35)
, suggesting that TWEAK might be an important mediator of inflammatory responses.
Although TWEAK and its receptor Fn14 are highly expressed in a variety of tissues, including skeletal muscle, the physiological or pathophysiological roles of this ligand-receptor dyad remain largely unknown. We recently showed that soluble TWEAK protein inhibits the differentiation of myoblasts into myotubes (36)
. However, the effects of TWEAK on differentiated skeletal muscle in vitro and in vivo have not yet been determined. Therefore, in the present study we have investigated the role and mechanisms by which TWEAK regulates skeletal muscle mass. Our results demonstrate that TWEAK is a strong muscle-wasting cytokine that induces degradation of muscle proteins through activation of the ubiquitin-proteasome and NF-B pathways. TWEAK also inhibits activation of the PI3K/Akt signaling pathway in skeletal muscle. Furthermore, chronic administration of soluble TWEAK protein or skeletal muscle-specific transgenic overexpression of TWEAK diminishes overall body weight and skeletal muscle mass in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, phospho Akt, phospho FKHR, phospho GSK3ß, phospho mTOR, and total Akt protein were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal antibodies against tropomyosin, ß-actin, and sarcomeric -actin were obtained from Sigma Chemical Company (St. Louis, MO, USA). Antibody against p52 protein was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Plasmid pNF-B-Luc was from BD Biosciences (San Diego, CA, USA). Rabbit polyclonal ubiquitin antibody was from CalBiochem (San Diego, CA, USA). Mouse monoclonal MF-20 antibody specific to myosin heavy chain-fast twitch (MyHCf) protein was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. AlexaFluor 546 goat anti-mouse antibody was purchased from Molecular Probes (Eugene, OR, USA). Protein A Sepharose beads were obtained from Pierce Biotechnology (Rockford, IL, USA). Recombinant mouse TWEAK, TNF- and TWEAK ELISA kit were from R&D Systems (Minneapolis, MN, USA). An annexin V-enhanced green fluorescence protein (EGFP) apoptosis detection kit was obtained from BioVision Inc. (Mountain View, CA, USA). NF-B consensus oligonucleotides and luciferase assay kit were purchased from Promega (Madison, WI, USA). Poly dI·dC was obtained from Amersham Biosciences (Arlington Heights, IL, USA). CK assay kit was obtained from Stanbio Laboratory (Boerne, TX, USA). [-32P] ATP (specific activity, 3000 Ci/mmol) was obtained from Perkin-Elmer Life Sciences (Boston, MA, USA).
Mice
Mice (strain: C57BL6) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed and fed in stainless steel cages on a 12 h on and 12 h off lighting schedule. Institutional Animal Care and Use Committee of the Jerry L. Pettis Memorial VA Medical Center, Loma Linda, approved experimental protocols. All procedures were conducted in strict accordance with public health service and animal welfare policy.
Cell culture
C2C12 (a myoblastic cell line) were obtained from American Type Culture Collection (Rockville, MD, USA). These cells were grown in DMEM containing 10% FBS. Primary myoblasts from neonatal mice were prepared by a method described previously (36
, 37)
. C2C12 and mouse primary myoblasts were differentiated into myotubes by incubation in differentiation medium (2% horse serum in DMEM) for 96 and 48 h, respectively. Myotubes were maintained in differentiation medium and medium was changed every 48 h.
Immunocytochemistry and myotube analysis
To study the effect of TWEAK on cultured myotubes, immunocytochemistry was performed as described previously (36
, 38)
. In brief, after treatment with TWEAK, myotubes were fixed with 3.7% paraformaldehyde, followed by permeabilization with 0.1% Triton-X-100. After being washed with PBS, the cells were blocked with 1% BSA in PBS for 1 h, then incubated with MF-20 antibody (specific to MyHCf protein) at 1:100 dilutions in PBS for 2 h. The cells were washed with PBS, incubated with goat anti-mouse IgG-Alexa 546 (2 µg/ml) for 1 h, and counterstained with 4',6'-diam idino-2-phenylidole (DAPI) for 5 min. Stained cells were analyzed under a fluorescent microscope (Olympus IX 70). Pictures were captured using Olympus MagnaFire Digital Camera and software. DAPI and MyHCf staining images were finally merged using MagnaFire software. We used Image Pro 4 software to measure myotube diameter. Myotube diameter was quantitated as follows: 10 fields were chosen randomly and 
10 myotubes were measured per field. The average diameter per myotube was calculated as the mean of the five measurements taken along the length of the myotube.
Construction and use of adenoviruses
Adenoviral vectors encoding human FLAG-IBN cDNA were constructed as described (39)
. Briefly, FLAG-IBN cDNA was isolated from pcDNA3-FLAG-IBN plasmid using HindIII and XbaI restriction enzymes and ligated at HindIII and XbaI sites into pAdTrack-cytomegalovirus (CMV) vector. Positive clones were linearized by the restriction endonuclease PmeI and cotransformed with the supercoiled adenoviral vector AdEasy-1 into Escherichia coli strain BJ5183 (Stratagene, La Jolla, CA, USA). Recombinant adenoviral constructs were selected, digested with restriction endonuclease PacI, and transfected into packaging cell line 293T using Effectene transfection reagent (Qiagen, Valencia, CA, USA). Production of adenovirus in 293T cells was observed after 6–7 days and was monitored by expression of GFP in viral plaques. The cells were collected 7–8 days after transfection; the adenoviruses were released by three freeze-thaw cycles and amplified by infecting 293T cells in one 100 mm tissue culture plate. After 3 days the adenoviruses were harvested as described above and further amplified by infecting 293T cells. The amplified adenoviruses were harvested 3 days later, purified by centrifugation in CsCl, and stored at –80°C in storage buffer [5 mM Tris-Cl (pH 8.0), 50 mM NaCl, 0.05% BSA, and 25% glycerol]. The titer of the virus was determined by infecting 293T cells with serial dilutions of adenoviruses and monitoring the viral plaques for expression of green fluorescence protein. C2C12 myoblasts (5x105 /well) were seeded in 6-well plates on day 0. On day 1, myoblasts were transduced with Ad.ß-Gal (kindly provided by Drs. M. E. Berens and N. L. Tran) or Ad.IBN at a multiplicity of infection (moi) of 500 particles/cell in 1 ml of growth medium and incubated for 1.5 h at 37°C, followed by addition of 1 ml of growth medium. After 24 h, the medium was changed with fresh growth medium or differentiation medium without adenoviruses. The expression of proteins in transduced myoblasts/myotubes was confirmed by Western blot.
Protein extraction, immunoprecipitation, and Western blot
C2C12 myotubes or skeletal muscle from mice were lysed in lysis buffer A (50 mM Tris-Cl [pH 8.0], 200 µM NaCl, 50 mM NaF, 0.3% Nonidet P-40, 2 mg/ml leupeptin, 2 mg/ml aprotinin, phenylmethylsulfonylfluoride, 0.5 mg/ml benzamidine, 1 mM dithiothreitol, and 1 mM sodium orthovanadate) and clarified by centrifugation described previously (40)
. Muscle extracts were normalized based on the amount of protein using the Bio-Rad (Hercules, CA, USA) protein assay reagent. Protein extracts (300 µg/ml) were immunoprecipitated with ubiquitin antibody (4 µg/sample). The immunoprecipitates were collected using protein A Sepharose beads, separated in 8% sodium dodecyl sulfate-polycrylamide gel (SDS-PAGE), transferred to nitrocellulose membrane, and blotted with MF-20 antibody. To check total or phosphorylated levels of the specific protein, 80 µg protein extracts from each sample were separated via 8–10% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the indicated antibodies as described (36
, 40)
. A 1:1000 dilution was used for all primary antibodies except MF-20, for which 1:100 dilution was used.
Creatine kinase (CK) assay
CK activity was measured using a spectrophotometric based kit as described (36)
. Specific activity of CK was calculated after correction for total protein and defined as units per milligram of protein (U/mg).
Electrophoretic mobility shift assay (EMSA)
NF-B activation was analyzed by EMSA as described earlier (36
, 40)
. Briefly, 10 µg of nuclear extract prepared from cultured myotubes or skeletal muscle tissue was incubated with 16 fmol 32P- ATP-end-labeled NF-B consensus double-stranded oligonucleotide for 20 min at 37°C. The incubation mixture included 2–3 µg of poly dI.dC in a binding buffer (25 mM HEPES [pH 7.9], 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl). The DNA-protein complex thus formed was separated from free oligonucleotides on a 7.5% native polyacrylamide gel. The gel was dried, and radioactive bands were visualized and quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) using ImageQuant software.
RNA Isolation and quantitative real time-polymerase chain reaction (QRT-PCR)
RNA isolation and QRT-PCR were performed by a method described previously (36
, 37)
. The sequences of primers used to measure the mRNA levels are as follows:
Atrogin-1: 5'-AAG GCT GTT GGA GCT GAT AGC A-3' (forward), and 5'-CAC CCA CAT GTT AAT GTT GCC C-3' (reverse); MuRF1: 5'-TGC CTG GAG ATG TTT ACC AAG C-3' (forward) and 5'-AAA CGA CCT CCA GAC ATG GAC A-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-ATG ACA ATG AAT ACG GCT ACA GCA A-3' (forward) and 5'-GCA GCG AAC TTT ATT GAT GGT ATT-3' (reverse). All reactions were carried out in triplicate to reduce variation. The data were analyzed using SDS software version 2.0 and results were exported to Microsoft Excel for further analysis. Data normalization was accomplished using GAPDH, and the normalized values were subjected to a 2–Ct formula to calculate the fold change between the control and experiment groups.
Generation of TWEAK transgenic mice
We used pCCLMCK-II vector (kindly obtained by Prof. Ulla Wewer), which drives the expression of the target cDNA using muscle creatine kinase (MCK) promoter (41)
. Full-length mouse TWEAK cDNA was ligated at the PacI site of the pCCLMCK-II vector. The insert and orientation of TWEAK cDNA in the vector were confirmed by automated DNA sequencing. The expression cassette DNA containing the MCK promoter, TWEAK cDNA, and poly(A) tail was excised by SwaI restriction enzyme and provided to the Core Transgenic Facility of the University of Southern California for injection into the pronucleus of a fertilized ovum. Founder mice obtained were screened for the presence of transgene by PCR using DNA from the tail tip of the mice. Increased expression of TWEAK in transgenic mice was confirmed by measuring TWEAK protein content in skeletal muscle extracts using a commercially available mouse TWEAK ELISA kit (R&D Systems).
Analysis of myofiber cross-sectional area
Transverse paraffinized muscle sections (8 µm) were prepared and subjected to hematoxylin and eosin (H &E) staining as recently described (40)
. Stained sections were visualized under Olympus IX 70 microscope and pictures were captured using Olympus MagnaFire digital camera and software. Fiber cross-sectional area was measured for 100 adjacent muscle fibers in each section for each mouse using Image Pro 4.0 software. In brief, the outline of each muscle fiber was manually marked using a "define the object" tool in the "measurements" parameter of the software; software then calculated the areas of the defined fiber using all the possible diameters and displaying the mean of the various areas.
Statistical analysis
Results are expressed as mean ± SD. The Student’s t test or ANOVA 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, myotubes in the TWEAK-treated cultures had a reduced mean diameter compared with the untreated cultures. Quantitative estimation from multiple fields of view further confirmed a significant reduction in average myotube diameter in TWEAK-treated cultures (Fig. 1B
). TWEAK treatment did not induce apoptosis in myotubes measured using the annexin V-EGFP apoptosis detection kit (data not shown). These data provide the initial evidence that TWEAK induces skeletal muscle atrophy in vitro.
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TWEAK stimulates degradation of muscle proteins in vitro
We then determined the in vitro effects of TWEAK on total protein content and the expression of specific muscle proteins. C2C12 myotubes were treated with increasing concentrations of soluble TWEAK protein for 72 h. At the end of the incubation period, all cells from each well were collected, cell extracts were made, and total protein content was measured. As shown in Fig. 2
A, TWEAK treatment significantly decreased the total protein content in C2C12 myotubes in a dose-dependent manner. We then determined the effect of TWEAK on the level of specific muscle proteins such as creatine kinase (CK), myosin heavy chain fast type (MyHCf), tropomyosin, and sarcomeric -actin. Treatment of C2C12 myotubes with TWEAK significantly reduced levels of CK (Fig. 2B
) and MyHCf without affecting the levels of tropomyosin and sarcomeric -actin (Fig. 2C
). These results are consistent with other published reports suggesting that only specific muscle proteins (not all) are degraded during muscle atrophy conditions such as in response to cytokines and/or tumor growth (42)
. Similar to C2C12 myotubes, TWEAK treatment also reduced levels of MyHCf protein in myotubes prepared from mouse primary myoblasts (Fig. 2D
). TWEAK did not affect the synthesis of MyHCf in cultured myotubes in our 35S-methionine pulse-chase experiments (data not shown).
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Since TWEAK and TNF- are structural homologues and TNF- is a widely known muscle-wasting cytokine (4
, 17
, 42)
, we also compared the effects of TWEAK and TNF- on degradation of MyHCf in C2C12 myotubes. At equimolar concentrations, TWEAK was more potent in inducing the degradation of MyHCf than TNF- (Fig. 2E
).
TWEAK augments activation of the ubiquitin-proteasome pathway and the expression of atrogin-1 and MuRF1 in cultured myotubes
To determine the underlying mechanisms responsible for skeletal muscle atrophy in response to TWEAK, we studied activation of the ubiquitin-proteasome system. Cultured C2C12 myotubes were treated with soluble TWEAK protein for different intervals ranging from 0 to 24 h; protein extracts were made, and an equal amount of protein was immunoprecipitated with ubiquitin antibody and immunoblotted with MyHCf antibody. As shown in Fig. 3
A, the level of ubiquitinated MyHCf was increased upon treatment of myotubes with TWEAK, indicating that TWEAK augments the activity of the ubiquitin-proteasome system.
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Among the hundreds of known E3 ubiquitin ligases, atrogin-1 and MuRF1 have recently been identified as inducible muscle-specific ubiquitin ligases that are involved in degradation of muscle proteins in a variety of conditions (3
, 5
6
7)
. We investigated whether TWEAK can induce the expression of atrogin-1 and MuRF1 in cultured myotubes. C2C12 myotubes were treated with TWEAK for different intervals and the mRNA levels of atrogin-1 and MuRF1 were determined by QRT-PCR technique. TWEAK caused an almost 3-fold increase in the mRNA level of atrogin-1 (Fig. 3B
) and 5-fold increase in the mRNA level of MuRF1 (Fig. 3C
) within 12 h. These data suggest that TWEAK-induced degradation of muscle protein occurs through the increased activation and expression of the ubiquitin-proteasome system components.
TWEAK inhibits activation of PI3K/Akt pathway in myotubes
Several recent studies have shown that inhibiting the activity of the PI3K/Akt pathway can lead to skeletal muscle atrophy and increase the expression of atrogin-1 and MuRF1 ubiquitin ligases (43
44
45)
. We examined whether TWEAK can affect the activation of PI3K/Akt signaling pathway in differentiated myotubes. C2C12 myotubes were treated with soluble TWEAK protein (10 ng/ml) for varying intervals and phosphorylation of the specific proteins was studied by Western blot using antibodies that recognize their phosphorylated form. The basal level of phosphorylation of Akt kinase was suppressed upon treatment of myotubes with TWEAK (Fig. 4
, top panel). Phosphorylation levels of the downstream target of Akt kinase such as GSK3ß, FOXO1a, mTOR, and p70S6K were also reduced in response to TWEAK treatment (Fig. 4)
. There was no effect on the total protein level of Akt, GSK3ß, FOXO1a, mTOR, or p70S6K upon treatment of myotubes with TWEAK (Fig. 4
and data not shown). These data suggest that TWEAK inhibits the activity of the PI3K/Akt signaling system in skeletal muscle.
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NF-B transcription factor is involved in TWEAK-induced degradation of muscle protein
Accumulating evidence suggests that activation of NF-B can lead to muscle atrophy especially in response to tumor growth and inflammatory molecules (46
47
48)
. We investigated whether TWEAK can activate NF-B in skeletal muscles and whether increased activation of NF-B is responsible for the degradation of muscle proteins in vitro. C2C12 myotubes were treated with 10 ng/ml TWEAK for different intervals and the activation of NF-B was measured by EMSA. As shown in Fig. 5
A (upper panel), treatment of C2C12 myotubes with TWEAK significantly increased the DNA binding activity of NF-B.
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NF-B can be activated through two parallel pathways. The canonical NF-B activation pathway requires the phosphorylation and degradation of NF-B inhibitory protein IB by upstream kinases (49)
. On the other hand, activation of the alternative NF-B pathway involves proteasome-mediated processing of the NF-B2 precursor p100 to produce p52 (50)
. We found that TWEAK-induced activation of NF-B involves phosphorylation of the IB protein. The level of phosphorylated IB protein, determined by Western blot, was significantly increased upon treatment of myotubes with TWEAK (Fig. 5A
, middle panel). However, there was no change in the relative levels of p100 and p52 upon treatment with TWEAK (Fig. 5A
, bottom panel). Furthermore, supershift analysis of NF-B/DNA complex seen in EMSA revealed that it contained mainly p50 and p65 subunits but not p52 (data not shown). Collectively, our data suggest that, in myotubes, TWEAK predominantly activates the classical NF-B signaling pathway.
We also measured NF-B transcriptional activity in C2C12 myotubes in response to TWEAK using a luciferase reporter assay. C2C12 myoblasts were transiently transfected with pNF-B-Luc plasmid (contains multiple kappa enhancer elements upstream of the luciferase gene) and differentiated into myotubes by incubation in DM for 96 h. Myotubes were then treated with 10 ng/ml TWEAK for 24 h and luciferase activity was measured using a luciferase assay kit and a luminometer. As shown in Fig. 5B
, the transcriptional activity of NF-B was significantly increased in response to TWEAK treatment.
To study the role of NF-B in TWEAK-induced muscle protein degradation, we determined the effect of the inhibition of NF-B on levels of MyHCf in cultured myotubes. C2C12 myoblasts were transduced with adenoviral vectors Ad.ß-gal or Ad.IBN (a dominant negative inhibitor of NF-B) for 24 h, followed by treatment with TWEAK and measurement of NF-B activity by EMSA. As shown in Fig. 5C
, overexpression of IBN protein inhibited the TWEAK-induced activation of NF-B. In another experiment, C2C12 myoblasts transduced with Ad.ß-gal or Ad.IBN were differentiated into myotubes by incubation in differentiation medium for 96 h, followed by treatment with different concentrations of TWEAK for 72 h. As shown in Fig. 5D
, the inhibition of NF-B through overexpression of IBN protein completely blocked the TWEAK-induced degradation of MyHCf. These data suggest that TWEAK activates NF-B and the increased activation of NF-B is responsible at least for the TWEAK-induced degradation of MyHCf in myotubes.
Chronic administration of TWEAK causes muscle atrophy in mice
The in vivo effects of TWEAK in mice were also investigated. Mice at the age of 3 wk were given either an intraperitoneally (i.p.) injection of soluble TWEAK (100 µg/kg body weight in 100 µl sterile saline solution) or saline solution alone every week for a total of 4 wk. We did not find any significant difference in either food or water uptake between TWEAK-treated and untreated groups of mice (data not shown). At the end of the protocol the mice were sacrificed, total body weight was recorded, and soleus and tibial anterior (TA) muscles were isolated tendon-to-tendon from left and right hind limb of the mice. Muscles were fixed in 10% formalin solution and embedded in paraffin; 5 µM sections were cut from the midbelly and stained with hematoxylin and eosin (H&E). The cross-sectional area of all of the myofibers of soleus muscle from individual mice was measured. Consistent with the in vitro data, in vivo administration of TWEAK significantly decreased overall body weight, soleus, and TA muscle weight (Fig. 6
A). Morphological analysis of muscle sections showed significantly reduced fiber diameter in TWEAK-treated mice compared with controls (Fig. 6B, C
).
|
In another experiment we investigated whether TWEAK can affect the activation of ubiquitin-proteasome system and NF-B in skeletal muscle in vivo. Mice were given a single i.p. injection of either PBS or PBS containing soluble TWEAK protein (100 µg/kg body weight). After 6 h, diaphragm and soleus muscles were isolated and processed for biochemical analysis. A significant increase in ubiquitination of MyHCf (Fig. 6D
) and activation of NF-B (Fig. 6E
) was observed in skeletal muscle of mice upon in vivo administration of TWEAK. These data suggest that similar to the cell culture system, increased activation of ubiquitin-proteasome system and NF-B might be responsible for TWEAK-induced degradation of muscle proteins in vivo.
Skeletal muscle-specific TWEAK transgenic mice have reduced body and skeletal muscle mass
We also investigated the effect of skeletal muscle-specific transgenic overexpression of TWEAK in mice. TWEAK was expressed in mice using the muscle creatine kinase (MCK) promoter. The design of the construct is shown in Fig. 7
A. In our first microinjection into the pronucleus of a fertilized ovum, we noted that the founder TWEAK transgenic mice were significantly smaller in size and died within 2 wk after birth, except one in which the copy number of transgene and expression of TWEAK were low. In the subsequent microinjections, we carefully monitored the body weight of the founder transgenic mice and littermate control mice after birth. Again, the size of the founder mice was smaller even at the time of birth and a few founder transgenic mice died with 1 wk of birth. At the age of 8 days, we took pictures and measured body weight of the entire surviving founder transgenic (n=5) and littermate control (n=21) mice. As shown in Fig. 7B
, TWEAK transgenic (Tg) mice at the age of 8 days looked smaller than the corresponding littermate control mice. Furthermore, the average body weight of the transgenic mice was significantly lower than the control mice (Fig. 7C
). We had to sacrifice two founder transgenic mice at the age of 8 days that had low body weight, abnormal breathing rate, and were highly fragile. Measurement of the TWEAK concentration in skeletal muscle extracts and serum using an ELISA assay kit showed a drastic increase in the TWEAK levels in skeletal muscle of founder TWEAK-Tg mice compared with littermate control mice (Fig. 7D
). Serum levels of TWEAK protein were also significantly (but not drastically) higher in TWEAK-Tg than in littermate control mice (Fig. 7D
). A gross comparison of lower hind limb showed that TWEAK transgenic mice had reduced muscle mass compared with littermate control mice (Fig. 7E
, upper panel). The size of the gastrocnemius muscle of transgenic mice was smaller than in littermate control mice (Fig. 7E
, lower panel).
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We also investigated whether the reduced muscle mass in TWEAK transgenic mice was associated with increased activity of the ubiquitin-proteasome pathway. Protein extracts made from gastrocnemius muscle of normal and TWEAK transgenic mice were immunoprecipitated with anti-ubiquitin rabbit polyclonal antibody (pAb) and blotted with MyHCf antibody. As shown in Fig. 7F
, ubiquitination of MyHCf was significantly higher in gastrocnemius muscle of TWEAK transgenic mice compared with littermate control mice. As expected, activation of NF-B was also higher in skeletal muscle of TWEAK transgenic mice (Fig. 7G
). Similar higher ubiquitination of MyHCf and activation of NF-B were also observed in skeletal muscle isolated from the carcass of the founder transgenic mice that died within 1 wk of birth. These data suggest that the reduced muscle mass in TWEAK transgenic mice could be due to increased activation of ubiquitin-proteasome and NF-B pathways.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B transcription factor. TWEAK-induced inhibition of the Akt signaling pathway might also contribute to the loss of skeletal muscle mass.
TWEAK was originally identified in 1997 by Chicheportiche et al. during a search for erythropoietin-related mRNAs in macrophages (23)
. Although TWEAK transcripts were detected in several tissues including brain, heart, skeletal muscle, pancreas, spleen, and peripheral blood monocytes (23)
, TWEAK knockout mice were viable, healthy, and fertile, suggesting that TWEAK is not crucial for normal development (31)
. However, recent studies have clearly shown that the expression of TWEAK and its receptor Fn14 is up-regulated in response to diverse stimuli and that TWEAK has pleiotropic effects on both immune and nonimmune cell types (26
, 29
, 31
, 51
52
53
54)
. Increased expression of TWEAK has also been observed in space-flown WI38 human fibroblasts (53)
and in several human tumor cell types (55
, 56)
that are known to cause muscle wasting. This further indicates that TWEAK might be an important mediator of skeletal muscle atrophy in different physiological and/or pathophysiological conditions.
Accumulating evidence suggests that the ubiquitin-proteasome system, which provides a mechanism for selective degradation of regulatory and structural proteins, is responsible for degradation of the bulk of muscle proteins in various atrophying conditions (2
, 57
, 58)
. It distinguishes damaged or misfolded proteins and labels the target proteins by conjugation with the polypeptide cofactor ubiquitin (59)
. Ubiquitin-conjugated proteins are then subsequently recognized by the 26S proteasome, a large multisubunit multicatalytic protease complex that degrades ubiquitinated proteins to small peptides (60)
. Although the upstream signaling events remain unknown, our data show that TWEAK rapidly increases the conjugation of ubiquitin to MyHCf (Fig. 3A
) and that ubiquitination preceded the degradation of MyHCf (Fig. 2C
and Fig. 3A
). Similar higher ubiquitination of MyHCf was also seen in skeletal muscle of TWEAK-treated mice (Fig. 6D
) and transgenic mice expressing TWEAK in skeletal muscles (Fig. 7F
). This evidence suggests that activation of the ubiquitin-proteasome system might be a major pathway that leads to increased proteolysis in skeletal muscle in response to TWEAK.
Even though the ubiquitin-proteasome system involves the concerted action of many proteins, the key enzyme in this system is E3 (ubiquitin ligase), which couples activated ubiquitin to lysine residues on the protein substrate and confers specificity to the system. Using transcriptional screening, two E3 ubiquitin ligases—atrogin-1 and MuRF1—were recently identified to be highly induced in the process of muscular atrophy (6
, 61)
. Increased expression of atrogin-1 and/or MuRF1 has been reported in animal models of muscular atrophy induced by immobilization, denervation, hind limb suspension, starvation, tumor load, lipopolysaccharide (LPS), and glucocorticoids (3
, 4)
. The catabolic role of atrogin-1 and MuRF1 has been supported by the fact that targeted deletion of atrogin-1 or MuRF1 showed reduced muscle atrophy in response to denervation and hind limb suspension (61)
. TWEAK treatment significantly increased mRNA levels of both atrogin-1 (Fig. 3B
) and MuRF1 (Fig. 3C
) in cultured myotubes and upon in vivo administration of TWEAK in mice (data not shown), further indicating that inducible expression of these E3 ligases might be responsible for TWEAK-induced muscle atrophy. Collectively, our data indicate that accelerated muscle loss in response to TWEAK involves activation of the components of the ubiquitin proteolytic system.
An interesting observation of the present study was that, in contrast to other muscle-wasting cytokines, TWEAK inhibited the phosphorylation of Akt kinase and its downstream targets in skeletal muscles (Fig. 4)
. Several recent studies have shown that inhibition of the PI3K/Akt pathway can lead to skeletal muscle atrophy (9)
. Pharmacological or molecular inhibition of Akt reduces the average diameter of myotubes in cultures (44)
. Akt1 and Akt2 knockout mice have reduced muscle mass compared with their normal counterparts (45)
. Conversely, activation of Akt kinase can prevent muscle loss in response to denervation (43
, 62)
. It has been suggested that activation of Akt increases skeletal muscle mass by activating anabolic and suppressing catabolic pathways. The phosphorylation of GSK-3ß by Akt releases its inhibitory effect on the translation initiation factor eIF2B (63
, 64)
. Akt phosphorylates and activates the mammalian target of rapamycin (mTOR) (62)
, which in its turn causes phosphorylation of p70S6K and PHAS-1/4E-BP1 (63
, 65)
. Phosphorylation of p70S6K and PHAS-1/4E-BP1 leads to the activation of pathways promoting protein synthesis and translation initiation, respectively. In addition, Akt is known to prevent muscle loss by phosphorylating the forkhead family of transcription factors such as FOXO1 and FOXO3a, which prevents their translocation from the cytosol to the nucleus (66)
. Recent studies have shown that atrogin-1 and MuRF1 are the transcriptional targets of FOXO transcription factors. Thus, activation of the PI3K/Akt signaling pathway in skeletal muscle inhibits the expression atrogin-1 and MuRF1 by inhibiting the activity FOXO1 and FOXO3a transcription factors (7
, 66
, 67)
. Our results demonstrating that, along with Akt kinase, the basal level of phosphorylation of GSK-3ß, mTOR, p70S6K, and FOXO1 was also diminished in TWEAK-treated myotubes (Fig. 4)
suggest that an inhibition of the PI3K/Akt pathway might contribute to skeletal muscle atrophy in response to TWEAK.
Prevailing evidence suggests that activation of NF-B can lead to muscle atrophy in conditions such as tumor growth, glucocorticoids, and cytokines (1
, 8)
. Recently, Cai et al. showed that muscle-specific transgenic expression of IB kinase ß (an upstream activator of NF-B pathway) causes profound muscle loss in mice and that skeletal muscle-specific inhibition of NF-B attenuated the denervation- and tumor-induced muscle loss in mice (46)
. Furthermore, atrophy resulting from hind limb unloading was blocked in nfkb1 and bcl3 gene knockout mice (47)
, suggesting that activation of NF-B subunits causes muscle atrophy in diverse conditions. As our data in Fig. 5
show, treatment of myotubes with TWEAK causes a rapid and sustained increase in both DNA binding and transcriptional activities of NF-B. These data are consistent with published reports from us (36)
and another group (35)
which suggest that TWEAK causes long-lasting activation of NF-B in several cell types, including myogenic cells. We found that TWEAK also activates NF-B in vivo in skeletal muscle of mice (Figs. 6
, 7)
. Since the inhibition of NF-B activity completely blocked the TWEAK-induced degradation of MyHCf in myotubes (Fig. 5D
), NF-B constitutes the pathways that are responsible for TWEAK-induced skeletal muscle atrophy. Although the exact mechanisms by which NF-B causes muscle atrophy remain enigmatic, there is a possibility that activation of NF-B augments the expression of certain genes involved in degrading muscle proteins. In fact, several genes, including MyoD (68)
, ubiquitin-conjugating E2 enzyme UbcH2/E220k (69)
, and proteasome C3 subunit, are targets of NF-B (70)
. In addition, NF-B seems to regulate the expression of MuRF1, which is also unregulated in response to TWEAK (Fig. 3B
). The promoter region analysis of mouse MuRF1 gene using online PromoterInspector software showed that it contains multiple NF-B binding sites (our unpublished data). The role of NF-B in regulating MuRF1 expression is further supported by a study demonstrating that transgenic expression of IKKß causes an almost 3-fold increase in the expression of the MuRF1 gene; muscle wasting in these mice was partially rescued by crossing them with MuRF1 knockout mice (46)
.
Our in vitro observations are well supported by in vivo studies, which demonstrate that chronic administration of soluble TWEAK protein in mice or transgenic overexpression of TWEAK in skeletal muscle reduces body and muscle weight in mice (Fig. 6
, Fig. 7
). Although more investigations are required to determine the mechanisms and type of muscle fibers TWEAK affects in vivo, it is possible that reduced muscle mass in TWEAK-treated mice or transgenic mice is a result of both TWEAK-induced muscle protein degradation, as supported by data in this study, and an inhibition of muscle regeneration, because TWEAK also inhibits differentiation of myoblasts into myotubes (36)
. We did not find any effect of TWEAK on the synthesis of MyHCf in cultured myotubes (data not shown). However, we cannot completely rule out the possibility that TWEAK might inhibit the synthesis of certain muscle proteins. Furthermore, since TWEAK is a secreted protein, reduced body weight of muscle-specific TWEAK transgenic mice could also be attributed to a general retardation in overall body growth. Indeed, the serum level of the TWEAK transgenic mice was higher than in control mice (Fig. 7D
). TWEAK may also interfere with the actions of growth factors during development and/or directly inhibit the activation of certain signal transduction pathways (e.g., PI3K/Akt) that are required for normal development (45)
.
The ongoing research in our laboratory further suggests that the actions of TWEAK in skeletal muscle are regulated by the expression of its receptor, Fn14. The activation of catabolic pathways such as NF-B in response to TWEAK was completely inhibited by knockdown of Fn14 receptor in myogenic cells using RNA interference technique (Dogra et al., unpublished results). The expression of Fn14 in skeletal muscle was higher only at the initial stages (up to 6 wk) of mouse development, which decreased to almost undetectable levels in older (3 months) mice (data not shown). More importantly, we found that skeletal muscle atrophy was prominent when TWEAK was administered in 3-wk-old mice (Fig. 6)
but not in 12-wk-old mice (data not shown). Similarly, the TWEAK transgenic mice that survived (due to low copy number of transgene) initially had lower body weights, but after 10–14 wk the body weight of normal and TWEAK transgenic mice was similar (data not shown). These observations clearly suggest that TWEAK regulates muscle mass by recruiting the Fn14 receptor and that the biological actions of TWEAK depend on the expression of Fn14 in skeletal muscle.
In summary, we provide the first evidence that TWEAK is a strong muscle-wasting cytokine. Although more investigations are required to determine the mechanisms that regulate TWEAK and Fn14 expression in skeletal muscle under different atrophying conditions, the findings in this study could be of high clinical significance because TWEAK might be the major mediator of skeletal muscle loss in many disease states, by itself or in combination with other catabolic molecules.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 18, 2006. Accepted for publication January 4, 2007.
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