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Department of Health Sciences, Boston University, Boston, Massachusetts, USA
1Correspondence: Department of Health Sciences, Boston University, Boston, Massachusetts, USA. E-mail: skandar{at}bu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: hind limb unloading • denervation • starvation • diabetes • ubiquitination
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Ubiquitin-protein ligases (E3s) catalyze the final step of substrate protein ubiquitination; the addition of ubiquitin moieties is a post-translational modification that marks a protein for degradation (3
, 4)
. Polyubiquitinated proteins are targeted for degradation by the 26S proteasome, whereas monoubiquitinated proteins are mainly degraded by lysosomal cathepsins (5)
. The high substrate specificity of E3s is due to their structural diversity and explains why there are many E3s in a given tissue (6)
. Consequently, the activation of multiple E3s during muscle atrophy results in the targeting and degradation of many different substrates that range in function from structural to signaling proteins. The activation of specific E3s then is a determinant of the atrophied muscle phenotype, which may differ depending on the triggering stimulus (7)
.
Several groups have identified E3s involved in skeletal muscle atrophy, including atrogin1/MAFbx (8
, 9)
, MuRF1 (9)
, and E3
ubiquitin-protein ligases (2
, 10)
, but there is a paucity of knowledge about the substrates of these E3s in muscle except MuRF1, which appears to target several myofibrillar and related structural proteins (11)
. Microarray data from our laboratory showed a significant increase in the ubiquitin-protein ligase called Nedd4 in atrophied soleus muscles from hind limb unloaded rats, and the magnitude of this increase was very similar to what was found for atrogin/MAFbx (12)
. Since our original finding, two other studies have shown increased Nedd4 expression in 14 days unloaded muscle (13)
and in 1 and 3 mo denervated muscle (14)
. Unlike atrogin/MAFbx or MuRF1 that are muscle specific and represent different types of RING finger containing ubiquitin ligases, Nedd4 is a homologous to E6-activating protein carboxyl terminus (HECT) domain containing ubiquitin ligase, and it is ubiquitously expressed. Besides its mRNA expression (12
13
14
15
16)
and in one case protein expression (14)
, Nedd4 has not been studied in skeletal muscle. In other tissues, it has a role in cellular processes involving turnover of membrane-bound channels and receptors associated with protein trafficking, endocytosis, virus budding, and transcription (17
, 18)
. Nedd4 associates with cellular membranes via its C2 lipid binding domain, allowing for the interaction between the WW domains of Nedd4 and PPXY motifs of its membrane-associated protein substrates (17
, 18)
.
The purpose of this study was to characterize expression of the ubiquitin ligase Nedd4 in different types of atrophy and in different muscle types and to identify a protein substrate of Nedd4 during muscle unloading atrophy. We first reconfirmed, using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the muscle unloading induced up-regulation of Nedd4 mRNA found in our microarray study (12)
. Next, because Nedd4 protein has not previously been localized to muscle fibers, we confirmed its localization. We also found that Nedd4 protein expression was increased in atrophy due to muscle unloading and denervation but not with food deprivation or diabetes, and so we sought to find a protein substrate of Nedd4 during unloading atrophy, as a clue to its differential expression. To do this, we first determined if known Nedd4 targets, identified in other cell types, were down-regulated in unloaded muscle. Of the eight Nedd4 targets we examined, only Notch1 was down-regulated in unloaded and denervated muscles, and, significantly, this was not observed in atrophied muscles from food-deprived or diabetic rats.
To identify Notch1 as a bona fide target of Nedd4, we showed that Nedd4 was both necessary and sufficient for the down-regulation of Notch1 with unloading atrophy in the soleus. To verify the targeting (i.e., ubiquitination) of Notch1 by Nedd4, conditional overexpression of Nedd4 in C2C12 muscle cells showed increased ubiquitination of Notch1. This is the first finding of a Nedd4 substrate in muscle and of an ubiquitin ligase the activity of which distinguishes disuse from cachexia atrophy. The present work sets the stage for determining the physiological implications of reduced Notch1 signaling during muscle atrophy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for 7 days. Denervation atrophy was induced by performing bilateral transection of the sciatic nerve. In the paired control group, a sham surgery was performed by exposing, but not transecting the sciatic nerve. The animals were killed 7 days postsurgery. Starvation atrophy was induced by withdrawing food but not water, from rats for 6 days. Muscle samples from diabetic rats were kindly provided by Dr. William Mitch (Baylor College of Medicine, Houston, TX, USA). Diabetes (insulinopenia) was induced by using streptozotocin (STZ) injection as described previously (20)
.
Muscle Preparation and Analysis
At the end of the experimental treatment, control and experimental rats were anesthetized using intraperitoneally injection of sodium pentobarbital (55 mg/kg body wt), and muscles from right and left hind limbs were removed, quickly weighed, and either processed immediately for RNA or protein assays or embedded in tissue freezing medium and frozen in isopentane for sectioning and subsequent morphological and immunohistochemical analyses.
Immunohistochemical Analysis
Muscles frozen in isopentane were sectioned (10 µm) from the midbelly and fixed in 4% paraformaldehyde. The sections were then blocked in 10% BSA in PBS for 1 h and incubated with rabbit anti-Nedd4 (Upstate Biotechnology, Lake Placid, NY, USA) and mouse MF20 (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA, USA) antibodies overnight at room temperature. Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) fluorescent dye conjugated to goat anti-rabbit IgG and Texas red (Molecular Probes) fluorescent dye conjugated to goat anti-mouse IgG were the secondary antibodies used for visualization. Images were visualized with a fluorescent microscope (Nikon, Melville, NY, USA) and captured with a Spot RT camera and Spot Software (Diagnostic Instruments, Sterling Heights, MI, USA). MetaMorph Imaging System (Molecular Devices Corporation, Sunnyvale, CA, USA) was used for calculating fiber cross-sectional areas.
Gene Transfer Using Intramuscularly DNA Injection
The T7 epitope-tagged Nedd4 constructs, pRC/cytomegalovirus Nedd4-T7 (21)
and catalytically inactive pRC/cytomegalovirus Nedd4(c
s)-T7 (22)
were gifts from Dr. Daniela Rotin at the University of Toronto. The enhanced GFP (EGFP)-c1 plasmid was purchased from Clontech (Palo Alto, CA, USA). Plasmid DNA injections into skeletal muscle have been previously detailed (23)
. Plasmid DNA was prepared using an endotoxin-free mega prep kit (Qiagen, Valencia, CA, USA) and suspended in PBS. Before plasmid injection, control and unloaded animals were anesthetized with sodium pentobarbital (55 mg/kg body wt). Rat soleus muscles were injected with 50 µg plasmid DNA in a volume of 40 µl. After injection, electric pulses were delivered, using an electric pulse generator (Electro Square porator extracellular matrix 830, BTX) by placing two paddle-like electrodes on each side of the muscle. Five electric pulses were delivered at 125 V/cm, a duration of 20 ms, and an interpulse interval of 200 ms. All rats were weight bearing for 1 days, and then one-half of the rats were hind limb suspended for 7 days. Hind limb unloading does not influence the efficiency of plasmid DNA uptake compared with weight-bearing control muscles (23)
. The retention of the plasmid and overexpression of the encoded protein are known to be stable for several months after injection (24
, 25)
.
Crude Membrane Isolation
Muscles were homogenized in buffer containing 10 mM Tris, 250 mM sucrose, 1 mM EGTA, 0.2 mM PMSF, and 100 U/ml protease inhibitor cocktail (Sigma, St. Louis, MO, USA) followed by centrifugation at 10,000 g for 20 min. The supernatant was brought to 0.6 M KCl final concentration with crystalline KCl and incubated on ice for 1 h. The sample was then centrifuged at 150,000 g for 45 min in an ultracentrifuge. The supernatant was decanted, and the centrifuge tubes were inverted to allow residual liquid to drain. The crude membrane pellet was resuspended in buffer containing 10 mM Tris (pH 7.5) with 0.1 mM PMSF and 100 U/ml protease inhibitor cocktail (Sigma).
Western blot Analysis
Muscle lysates were prepared by homogenization in 1x lysis buffer (Cell Signaling, Danvers, MA, USA) and centrifuging at 5,500 g for 20 min, and the supernatant was used as lysate. Protein concentrations for both crude membrane isolates and whole muscle isolates were determined using the DC protein assay (Bio-Rad, Hercules, CA, USA). Tweny micrograms of protein from muscle lysates were denatured in SDS loading buffer, boiled, and centrifuged briefly to remove insoluble material and separated on 4–20% SDS-polyacrylamide gels. Protein was transferred onto immobilon-FL polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Membranes were blocked in 5% nonfat milk for 1 h and incubated for 1 h to overnight with the appropriate primary antibody (Ab) diluted according to the manufacturer. Alexa Fluor 680 (Invitrogen, Carlsbad, CA, USA) or IRDye800 (Rockland Immunochemicals, Gilbertsville, PA, USA) fluorescent dye conjugated secondary antibodies were used for visualization with an Odyssey system (Li-Cor Biosciences, Lincoln, NE, USA). After the proteins of interest were visualized and the images were recorded, the PVDF membranes were incubated in Ponceau S protein staining solution (Sigma) for 10 min followed by destaining in water for 2 h. The total protein on the membrane was then visualized using the Odyssey system to confirm approximate equal protein in each lane of each blot (Li-Cor Biosciences). In addition, protein expression levels of ENaC, PKC-
, and Bcl-10, which did not change in response to any of the experimental conditions, were used for normalization of Nedd4 and Notch1 protein expression.
Cell Culture and Transfections
Approximately 1.2 x 106 mouse myoblast C2C12 (American Type Culture Collection, Rockville, MD, USA) cells were plated on 100 mm polystyrene plates with Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen), supplemented with 10% FBS (FBS; Invitrogen) and 1% penicillin/streptomycin (Invitrogen) and grown overnight at 37° in 5% CO2. The next day the cells were transfected with plasmid DNA, using Effectene transfection reagent (Qiagen) in differentiation media (DMEM supplemented with 2% horse serum and 1% penicillin/streptomycin) for 48 h. For conditional over-expression of Nedd4, the Tet-On system was used (Clontech). We created the target vector, pBI-enhanced GFP-Nedd4, by inserting the Nedd4 cDNA from pRC/cytomegalovirus Nedd4-T7 into pBI-enhanced GFP (Clontech) using HindIII. The cells were cotransfected with pBI-enhanced GFP-Nedd4, pMCK-rt-TA (a gift from Dr. Parker Antin, University of Arizona, Tucson, AZ, USA), and pMT123, the hemagglutinin (HA)-tagged polyubiquitin (8x Ub) expressing plasmid (kindly provided by Dr. Dirk Bohmann, University of Rochester, Rochester, NY, USA). At day 4 of differentiation, the cells were treated with doxycycline (1 mg/ml, Sigma), which allows binding of the rev tet transactivator (expressed by the pMCK-rt-TA plasmid) to the TRE in the pBI-enhanced GFP-Nedd4 plasmid, thus inducing expression of both EGFP and Nedd4.
Coimmunoprecipitation
At day 7 of differentiation, C2C12 cells were lysed using 1x cell lysis buffer (Cell Signaling) and protein concentrations were determined using the DC protein assay (Bio-Rad). Immunoprecipitation was performed using the Catch and Release kit (Upstate) according to the manufacturer’s instructions. Anti-hemagglutinin tag Ab (Upstate) was added to 2 mg of the whole cell lysates and incubated overnight at 4°C. After the washes, samples were eluted with denaturing buffer, boiled for 5 min, separated on a SDS-polyacrylamide gel, and processed for Western blotting.
RNA Isolation and RT-PCR
Total RNA was isolated from rat soleus muscles using a Trizol-based protocol (Invitrogen). A second "clean-up" step was used to improve the quality of total RNA using columns from Qiagen. RNA quality was judged based by the ratio of absorbance at 260 and 280 nm and quantitated at 260 nm. RNA samples were analyzed using agarose gel electrophoresis and stained to check for integrity of 18S and 28S RNA. cDNA was synthesized using 1 µg total RNA in a 20 µl reaction using RETROscript first strand synthesis kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The cDNA (2 µl) was then used as a template in a PCR reaction using specific primers for Nedd4 (GenBank accession no. XM_486230) and 18S (GenBank accession no. X01117) that were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/index.html) or using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primers for PCR amplification were as follows: Nedd4, forward, 5'-GTGGGAAGAGAGGCAGGATGTC-3', and reverse, 5'-GCGAATTCACAGGAAGTGTAGGC; and 18S, forward, 5'-CGCGGTTCTATTTTGTTGGT-3', and reverse, 5'-AGTCGGCATCGTTTATGGTC-3'. Once the conditions were optimized for each primer pair, PCR was performed and the amplification products were run on a 10% acrylamide gel and stained with SYBR Green (Molecular Probes), and densitometry was performed using the Alpha Innotech Alpha Imager 2000 imaging system.
Statistics
A Student’s t test was used to determine statistical significance between each control and experimental group. All data are expressed as means ± SE, and significance was established at the P < 0.05 level. The number of muscles studied for each experiment is given in the figure legends.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nedd4 localization to muscle cells has not previously been shown in whole skeletal muscle. Using immunohistochemical analysis, we demonstrated that Nedd4 protein is expressed in skeletal muscle and is localized to the sarcolemmal region in cross-sections of rat soleus muscles (Fig. 1
A). The expression of Nedd4 by cells in the intracellular space cannot be ruled out since the Nedd4 fluorescence signal also appears there. Crude muscle membrane preps showed high expression of Nedd4 further implicating its sarcolemmal localization, as shown below. A confirmation that Nedd4 protein is expressed in muscle cells is the high expression levels found in a mouse myogenic cell line (C2C12) in comparison to rat kidney lysates (Fig. 1B
), which are known to express Nedd4 (21)
.
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Hind limb unloading causes up-regulation of Nedd4 mRNA levels
We reconfirmed our previous findings (with microarrays) showing up-regulated Nedd4 mRNA at 1, 4, 7, and 14 days of unloading (12)
in our current study using RT-PCR (Fig. 2
). Nedd4 mRNA levels were significantly up-regulated at all time points. Samples from the 4 and 7 day hind limb unloaded muscles expressed at least 2-fold more Nedd4 mRNA than from age-matched weight-bearing control muscles (Fig. 2B
).
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Hind limb unloading induces increased expression of Nedd4 protein
In contrast to the soleus muscle, the tibialis anterior (TA), located in the anterior compartment of the lower hind limb, does not atrophy when unloaded (Fig. 3
A). However, 7 days of hind limb unloading resulted in a 2-fold increase of Nedd4 protein levels by in rat soleus homogenates and in crude membrane preparations (Fig. 3B, C
). Nedd4 protein expression in the membrane fraction supports the data showing sarcolemmal localization of Nedd4. The fact that Nedd4 was not increased in unloaded TA muscles, which do not atrophy, showed that the up-regulation of Nedd4 protein in soleus muscles is an atrophy-associated response rather than a part of a systemic response to hind limb suspension (Fig. 3B, C
).
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Increased Nedd4 protein expression only occurs in disuse (unloading and denervation) atrophy but not in starvation or diabetes atrophy
To establish if Nedd4 up-regulation is specific to unloading, we also measured its expression in atrophied soleus muscles of sciatic nerve transected (denervated), starved, and diabetic rats (Fig. 4
A). Nedd4 was up-regulated in denervated (and unloaded) soleus muscles by 2-fold, but there was no change in expression with starvation and diabetes (Fig. 4B
). Gastrocnemius muscles also atrophied due to unloading, denervation, starvation, and diabetes (Fig. 5
A), but again, Nedd4 protein was significantly up-regulated only in unloaded and denervated muscles by 42 and 86%, respectively (Fig. 5B
). Plantaris muscles atrophied in response to unloading, denervation, and starvation, but Nedd4 protein expression was increased only in unloaded and denervated muscles by 35 and 78%, respectively (see supplemental data). Diabetic plantaris muscles were not available for study. For the plantaris and gastrocnemius muscles, the greater atrophy with denervation compared to unloading was associated with a bigger decrease in Nedd4 expression.
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Identification of candidate Nedd4 target proteins
There are several targets of Nedd4 that have been identified in nonmuscle (18
, 22
, 27
28
29
30
31
32
33
34)
. To identify potential targets of Nedd4 in atrophied skeletal muscle, protein expression of known Nedd4 substrates was measured. There was no decrease in expression of ENaC, IGF1-R, Bcl-10, Eps15, vascular endothelial growth factor (VEGF)-R2, PKC-, and PLC-
1 in the unloaded and denervated muscles (see supplemental data). However, we did find that expression of full-length Notch1 (Notch1 FL) and its cleaved, activated intracellular domain (Notch1 ICD) were significantly decreased (30–40%) in soleus muscles with unloading and denervation but not with starvation and diabetes (Fig. 4C, D
). As with soleus muscles, gastrocnemius muscles showing up-regulation of Nedd4 (Fig. 5B
) also showed significantly decreased Notch1 ICD expression (Fig. 5C
). Tibialis anterior muscles that do not atrophy or show increased Nedd4 expression with unloading (Fig. 3A, B, C
) also did not show a change in Notch1 ICD expression (data not shown).
Nedd4 is required for hind limb unloading-induced down-regulation of Notch1
After establishing that increased expression of Nedd4 protein was correlated with decreased expression of one of its target proteins, Notch1, we tested whether the increase in Nedd4 is required for the down-regulation of Notch1 in unloaded soleus muscles. Electrotransfer of a dominant-negative Nedd4(cs) plasmid into soleus muscles of unloaded rats was used to inhibit the activity of endogenous Nedd4. Nedd4(cs) is a catalytically inactive form of Nedd4 carrying a loss of function mutation in its HECT ubiquitin-protein ligase domain (22)
. This form of Nedd4 is still able to bind its targets via its WW domains but lacks the ability to ubiquitinate them, thereby preventing the endogenous Nedd4 from associating with these substrates. Direct injection of Nedd4(cs) plasmid into soleus muscles completely rescued the unloading-induced down-regulation of Notch1 (Fig. 6
), suggesting that Nedd4 activity is required for the down-regulation of Notch1.
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The electrotransfer of Nedd4 plasmid DNA results in the injected plasmid being taken up only by the muscle cells and not by other cell types present in muscle, such as endothelial cells and fibroblasts (24
, 25)
. Since Notch1 levels were affected by electrotransfer of Nedd4 plasmid DNA, this is further evidence that Nedd4-regulated Notch activity occurs in muscle fibers.
Nedd4 is sufficient to induce down-regulation of Notch1 in weight-bearing rats
To determine whether overexpression of Nedd4 alone results in decreased levels of Notch1, we injected either EGFP or Nedd4 expression plasmids into soleus muscles of weight-bearing rats and assessed Notch1 expression (Fig. 7
). Muscles overexpressing Nedd4 showed statistically lower levels (30–40%) of Notch1 FL (Fig. 7B
) and Notch1 ICD (Fig. 7C
), suggesting that Nedd4 alone is sufficient to down-regulate Notch1 in soleus muscles of weight-bearing rats.
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Nedd4 overexpression leads to increased Notch1 ubiquitination in C2C12 myotubes
Since Nedd4 appears to target Notch1 during unloading atrophy, we tested if overexpression of Nedd4 increases ubiquitination of Notch1. Coimmunoprecipitation was used in C2C12 cells overexpressing HA-tagged polyubiquitin and conditionally overexpressing Nedd4 (Fig. 8
). The Tet-On conditional overexpression system was used to ensure overexpression of Nedd4 only on addition of doxycycline (Dox) to the mature myotubes, therefore, preventing overexpression of Nedd4 during differentiation. Two days after Dox treatment of C2C12s, only the lysates of C2C12s that received the Dox showed expression of exogenous Nedd4 (Fig. 8A
). Cells overexpressing Nedd4 were characterized by increased levels of ubiquitin-Notch1 complexes as compared to the cells overexpressing EGFP alone (Fig. 8B, C
). Cells that are transfected with one plasmid are also transfected with other plasmids in the transfection mixture. Thus expression of HA-polyubiquitin and Nedd4 was an enrichment strategy so that immunoprecipitation of HA-polyubiquitin selected proteins only from the population of cells that also overexpressed Nedd4. Subsequent immunoblotting with anti-Notch1 showed that it was bound in a complex with polyubiquitin. These data suggest that overexpression of Nedd4 alone ubiquitinates Notch1 in C2C12 myotubes.
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Nedd4 overexpression and fiber size
Although we did not find Nedd4 localized to the compartment of the myofibers where it would be expected to target myofibrillar proteins, we nevertheless determined its association with muscle fiber atrophy. As expected, inhibition of Nedd4 by overexpression of Nedd4(cs) did not inhibit unloading induced atrophy of soleus muscle fibers at 7 days (data not shown). In addition, overexpression of WT Nedd4 was not sufficient to induce a reduction in fiber cross-sectional area in weight-bearing rats (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 36)
. This finding generated the interest for studying components of different proteolytic pathways in atrophying muscles (37
38
39
40
41)
. Several studies have revealed the central role of the ubiquitin-proteasome pathway in atrophy (42
43
44)
. For the most part, substrate specificity-determining components of the ubiquitin-proteasome system are the E3 ubiquitin ligases. E3s ubiquitinate specific substrates, which are then degraded by the lysosome or the proteasome (6)
. Atrogin1/MAFbx1, MuRF1, and E3 are E3s, the functions of which have been most often studied during skeletal muscle atrophy (10
, 11
, 45
46
47
48)
. These ubiquitin ligases are up-regulated in multiple types of atrophy, but their target proteins are not well characterized except for MuRF1, which seems to have specific myofibrillar and structural proteins as targets (11)
.
Nedd4 is a HECT domain containing E3 ubiquitin-protein ligase that is expressed in skeletal muscle (15
, 16)
. In a microarray time course (1, 4, 7, and 14 days) analysis of unloading atrophy, we found a striking up-regulation in Nedd4 (12)
. Since our report of Nedd4 mRNA up-regulation with unloading, two other laboratories showed the same result with long-term atrophy; 14 days of unloading (13)
and 1 and 3 mo of denervation (14)
. Work on the localization of Nedd4 or of the protein substrates of Nedd4 in skeletal muscle has not been reported. Here we demonstrate that Nedd4 protein is expressed in skeletal muscle cells and that it localizes to the sarcolemmal region of adult skeletal muscle fibers, similar to the plasma membrane localization in other cell types and consistent with the lipid binding domain of Nedd4 (16
, 21)
. We found a significant up-regulation of Nedd4 protein expression in the atrophied muscles (soleus, gastrocnemius, and plantaris) of unloaded and denervated rats but not in starved or diabetic rats, suggesting for the first time, an ubiquitin ligase the activity of which distinguishes different types of atrophy. The trigger that leads to the up-regulation of Nedd4 in disuse atrophy but not in starvation or diabetic atrophy is not known but appears to relate to the loss of muscle tension. Perhaps it involves integrin signaling (49)
or the more recently discovered titin mechanotransduction (50)
.
Importantly, the substrates of Nedd4 in skeletal muscle have not been investigated. Of eight known Nedd4 targets found in other cell types, we found that only Notch1 was significantly down-regulated in unloaded atrophied muscles and thus was a candidate target protein of Nedd4 during unloading atrophy. Notch1 was then found to be a bona fide target of Nedd4 because Nedd4 was both necessary and sufficient for Notch1 down-regulation due to unloading-induced atrophy of the soleus. That is, genetic inhibition of Nedd4 protein, by use of a dominant negative Nedd4, completely inhibited the down-regulation of Notch1 in unloaded soleus muscles, and also, WT Nedd4 overexpression was sufficient to induce down-regulation of Notch1 receptor in the muscles of normal weight-bearing animals. The C2C12 experiments where Nedd4 and HA polyubiquitination were overexpressed indicated that Nedd4 is responsible for the ubiquitination of Notch1, further identifying Notch1 as a Nedd4 substrate.
On binding by the ligands Delta, Serrate, or Jagged, the full-length 300 kDa Notch1 receptor (Notch1 FL) undergoes partial proteolytic processing leading to the release of the "activated" 110 kDa intracellular domain (Notch1 ICD), which translocates to the nucleus and regulates transcription of target genes (51
, 52)
. This ligand-dependent activation of Notch1 ICD and subsequent activation of its target genes, such as Hes family of proteins, directs cells to remain in an undifferentiated state (51)
and also appears to be required for the activation of satellite cell proliferation in muscle regeneration (53)
. In contrast to this partial proteolysis and activation of Notch1 FL, it can also be targeted for complete proteolysis in a ligand-independent manner, thereby preventing transactivation of its targets (54)
. Nedd4 is responsible for the ubiquitination of several receptors and channels, including Notch1, which leads to their internalization and degradation mainly via the endocytotic lysosomal pathway (18
, 55)
. Protein-protein interaction (WW) domains of Nedd4 are known to have high affinity for PPXY motifs of target proteins (56
, 57)
. Two groups have independently identified Notch1, which contains multiple PPXY motifs in its intracellular domain, as a Nedd4 target during Drosophila wing development. Here, Nedd4 antagonizes Notch1 signaling by ubiquitinating it and marking it for internal degradation (33
, 34)
; this is similar to the findings in the present study showing decreased expression of Notch FL and Notch ICD in unloaded and denervated soleus muscle. The assessment of both FL and ICD was a way to distinguish ligand-independent degradation of the Notch receptor vs. ligand-dependent activation of Notch, which would have shown an increased ICD compared to FL Notch.
The specific role of Notch signaling in adult muscle is not well understood. Since most of the known roles of Notch signaling are associated with the developmental processes of proliferation and differentiation, the focus of Notch studies in adult muscle has been associated with investigating its role in satellite cells during muscle remodeling and regeneration. Notch signaling appears to be required for satellite cell proliferation after muscle injury, although this signal must later be controlled in order for the new myoblasts to differentiate into myotubes (53)
. Muscles of aged animals have a diminished regenerative potential compared to muscles of young animals. This has been attributed to the impaired proliferative capacity of satellite cells from aged muscles rather than to a decrease in their number (58)
. Forced Notch activation in aged muscle restored the proliferation capacity of satellite cells and therefore muscle regeneration, suggesting that Notch signaling plays a key role in the muscle regenerative potential that declines with age (58)
. The activation of Notch1 in muscle regeneration appears to involve the up-regulation of Delta, a Notch1 ligand (53)
. However, atrophy is not associated with muscle regeneration or activation of satellite cells; in fact, there is a loss of satellite cells in unloading (59)
. We measured Delta1 protein expression in our muscles using immunoblotting, and it was not detectable either before or after 7 days of unloading in agreement with its lack of expression in nonregenerating muscle (53
, 60)
(data not shown). This further supports the idea that Nedd4 participates in ligand-independent Notch regulation. Perhaps the decrease in Notch signaling in disuse atrophy has a role in the reduction of proliferative activity and number of satellite cells. Muscle reloading after unloading is accompanied by injury and regenerative processes (61
, 62)
. Therefore, experimental inhibition of Nedd4 might lead to an improved recovery process after unloading, via the maintenance of Notch1 signaling and more robust satellite cell activity.
Although ubiquitin ligases are fundamentally involved in the increased protein degradation associated with different types of atrophy, the knockout or inhibition of only one E3 may not block atrophy unless it has significant myofibrillar targets or if it targets a protein required for the regulation of protein degradation. For instance, E3 null mice did not show any effect on fasting-induced atrophy (63)
, yet a large body of work exists on the role of this ligase in atrophy (2)
. Knockout of MuRF1 gene did not affect the extent of denervation atrophy at 7 days, but it attenuated atrophy by 36% at 14 days (9)
. Atrogin/MAFbx knockout mice showed 25 and 56% inhibition of denervation atrophy at 7 and 14 days, respectively (9)
. With the exception of atrogin1, the small effect on atrophy due to the knockout of a given E3 is likely due to the fact that each ubiquitin ligase has relatively few substrates, which may not be major myofibrillar proteins or proteins that regulate degradation. The targets of some muscle specific ubiquitin ligases are just beginning to be elucidated (11
, 45)
. It is not surprising, therefore, that expression of dominant negative Nedd4 in soleus muscles did not block unloading atrophy at 7 days. This may be due to the fact that Nedd4 targets membrane proteins that do not affect gross fiber size. Therefore, Nedd4 targeting of Notch1 likely has a regulatory function during disuse atrophy, perhaps related to the functions discussed above.
In conclusion, we have defined, for the first time, the up-regulation of a specific ubiquitin ligase that distinguishes disuse from cachexia atrophy and, importantly, that Notch1 is a bona fide target of the ubiquitin ligase Nedd4 during unloading muscle atrophy. The present work sets the stage for determining the physiological role of reduced Notch1 signaling in atrophying muscle such as testing whether Notch1 plays a role in the recovery from unloading atrophy (e.g., reloading), which is characterized by limited injury and regeneration.
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ACKNOWLEDGMENTS |
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Received for publication June 12, 2006. Accepted for publication September 22, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 E. E. Dupont-Versteegden, R. Nagarajan, M. L. Beggs, E. D. Bearden, P. M. Simpson, and C. A. Peterson Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1263 - R1273. [Abstract] [Full Text] [PDF]
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Significantly different from EGFP injected unloaded muscles (P<0.05).
