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Department of Medicine, Baylor College of Medicine, Houston, Texas, 77030 USA
Correspondence: Aladin M. Boriek, Department of Medicine, Pulmonary Division, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: boriek{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B) pathways in skeletal muscles from normal and mdx mouse, a model of Duchenne muscular dystrophy (DMD), were investigated. The NF-B/DNA binding activity of the diaphragm muscle was increased by the application of axial mechanical stretch in a time-dependent manner. The increased activation of NF-B was associated with a concomitant increase in I-kappaB (IB) kinase activity and the degradation of IB
protein. Pretreatment of the muscles with nifedipine (a Ca2+ channel blocker) and gadolinium(III) chloride (a stretch-activated channel blocker) did not alter the level of activation of NF-B, ruling out involvement of Ca2+ influx through these channels. Furthermore, N-acetyl cysteine, a free radical inhibitor, blocked the mechanical stretch-induced NF-B activation, suggesting the involvement of free radicals. Compared with normal diaphragm, the basal level of NF-B activity was higher in muscles from mdx mice, and it was further enhanced in mechanically stretched muscles. Furthermore, activation of NF-B and increased expression of inflammatory cytokines IL-1ß and tumor necrosis factor in the mdx mouse precede the onset of muscular dystrophy. Our results show that mechanical stretch activates the classical NF-B pathway and this pathway could be predominately active in DMD.
Key Words: mechanical loading • TNF- • IL-1ß • free radicals
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B), a transcription factor, regulates the expression of a plethora of genes, especially those involved in the inflammatory and acute stress responses (1)
. NF-B is maintained in an inactive form in the cytoplasm through its interaction with the inhibitory proteins I-kappaB (IB). IB possesses both a nuclear localization signal and a nuclear export sequence that allow it to shuttle between the cytoplasm and the nucleus. Proteolytic degradation of IB upon its phosphorylation liberates NF-B to enter the nucleus and control the NF-B-regulated target genes (2)
. Recently, the kinase complex known as IB kinase (IKK) that phosphorylates the IkB protein has been identified (3)
. IKK is composed of two catalytically active subunits: IKK, IKKß, and a regulatory subunit IKK
. Although IKK and IKKß share a high degree of similarity in sequence and substrate specificities, their physiological functions are quite distinct. IKK plays a central role in keratinocyte differentiation but is not required for cytokine-dependent activation of NF-B. In contrast, IKKß is essential for NF-B activation by proinflammatory stimuli (4
, and references therein).
The role of NF-B in the process of skeletal muscle wasting is gaining increasing attention mainly because NF-B is activated in response to several inflammatory molecules that cause muscle loss (5
, 6)
. Elevated levels of circulating tumor necrosis factor- (TNF-), interleukin-1ß (IL-1ß), and matrix metalloproteinases (MMPs) have been observed in different types of muscular dystrophy (7
8
9
10)
. Although activation of a detrimental signal transduction pathway has been suggested as a probable cause for muscular dystrophy, the molecular mechanism that leads to an increased expression of these molecules in muscular dystrophy remains obscure. Recently, Hunter et al. provided evidence that unloading of the rat skeletal muscle that causes muscular atrophy activates the alternative NF-B pathway that requires the activation of IKK (11)
. However, the effect of mechanical loading, which generally leads to muscle hypertrophy, on the regulation of NF-B pathways remains unknown.
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease leading to early disability and to death, usually in early adulthood (12)
. The disease results from the absence of the structural protein dystrophin, which provides a structural base for the assembly of an integral membrane protein complex (13
14
15)
. The lack of dystrophin in the mdx mouse, a model for DMD, has been shown to severely compromise the mechanical integrity of myofibers and may cause the muscle fibers to be more vulnerable to mechanical stresses, hypo-osmotic shock, and contraction-induced damages (16
17
18)
. Furthermore, an aberrant activation of c-jun-N-terminal kinase1 (JNK1) has been previously reported in mdx mice that contributes to the dystrophic muscle pathogenesis (19)
. The activation of mitogen-activated protein (MAP) kinase signaling pathways generally leads to an altered gene expression by modulating the activity of several transcription factors, including the NF-B (20)
. However, it is not known whether the activity of NF-B is affected in the mdx mice and whether mechanical stretch modulates the activation of NF-B in the mdx mice.
In this study, we determined the ex vivo effects of mechanical stretch of skeletal muscles on the activation of NF-B pathways in normal and mdx mice, using mechanical testing and biochemical techniques. Our results demonstrate that 1) mechanical stretch of skeletal muscle activates the canonical NF-B pathway, possibly through the generation of free radicals; 2) skeletal muscle-specific NF-B activation and the expression of TNF- and IL-1ß precede the onset of muscle dystrophy in mdx mice; and 3) mechanical stretch of the diaphragm from mdx further activates the NF-B pathway signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, p50, p65, IKKß, JNK1, Raf1, ß-actin, and mutated NF-B consensus oligonucleotides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). nifedipine, gadolinium(III) chloride, and N-acetyl cysteine (NAC) were purchased from Sigma (St. Louis, MO). TRIzol reagent, oligo (dT)12–18 primer, reverse transcriptase, and Taq DNA polymerase were from Invitrogen Life Technologies (Carlsbad, CA). NF-B consensus oligonucleotide was obtained from Promega (Madison, WI). GST-IB (1–54) constructs was kindly provided by Dr. T.H. Tan. Poly dI·dC was from Amersham Biosciences (Arlington Heights, IL). 32P--ATP (specific activity, 3000 (111 TBq) Ci/mmol) was obtained from Perkin-Elmer (Boston, MA).
Mice and tissue preparation
Mice (C57BL6/SCSN and DMDMDX/J) were purchased from Jackson Laboratory, Bar Harbor, ME. These mice were housed and fed in stainless steel cages on a 12 h on and 12 h off lighting schedule. The animal facility is a virus-free facility. Experimental protocols were approved by the Animal Protocol Review Committee of the Baylor College of Medicine Animal Program (animal welfare assurance no. A-3823–01) and are assigned protocol # AN-1727. All procedures were conducted in strict accordance with public health service animal welfare policy. To study the effects of mechanical stress on NF-B activation in normal mice, we used the 129S1/SvImJ strain of adult mice (170 ± 12 days) with average body weight 23 ± 1.8 g. To study the activation of NF-B in mdx mice, we used DMDMDX/J (mdx) and C57BL6/SCSN (control) mice in four to five age groups. The control mice that were 15, 18, 23, 30, and 60 days old had average body weight of 5.4 ± 0.8, 6.9 ± 1.2, 8.3 ± 0.7, 13.2 ± 1.4, and 17.2 ± 1.7 g, respectively, whereas the mdx mice of the same age weighed 5.6 ± 0.6, 6.8 ± 0.5, 9.4 ± 0.9, 14.6 ± 0.8, and 20.3 ± 1.3 g. Mice were anesthetized with an i.v. injection of pentobarbital (0.5–0.7 ml/kg). The diaphragm muscle was excised from each animal and immediately immersed in a muscle bath containing a modified Krebs-Ringers solution (in mM: 137 NaCl, 5 KCL, 1 NaH2PO4, 24 NaHCO3, 2 CaCl2, 1 MgSO4; pH 7.4) bubbled with 95% O2/5%CO2. To study the effects of Gd3+ ions on NF-B activation, we used HEPES buffer (in mM: 10 HEPES [pH 7.4], 137 NaCl, 5 KCL, 2 CaCl2, 1 MgSO4). The solution was maintained at a temperature of 25°C throughout the muscle preparation and experimental mechanical testing phase of this study.
Ex vivo mechanical stretch of skeletal muscles
We used a mechanical testing apparatus to apply mechanical stress in the plane of the muscle sheet. The mechanical testing apparatus has two perpendicular axes, each driven by a micrometer. Two small identical clamps, one for each side of the muscle, were used to hold the muscle during mechanical stretch. We mechanically tested three muscles: diaphragm, abdominal wall, and biceps femoris. The detailed description of this apparatus is described previously (21)
. For the diaphragm, the muscle was secured in the muscle bath by fixing one tissue clamp on the central tendon and the opposing clamp on the myotendinous junction (MTJ) at the insertion on the rib cage. For the abdominal wall, the muscle was secured by fixing opposing clamps on the muscle near cartilaginous tissue. For the biceps femoris, the muscle was secured by fixing opposing clamps on the muscle near the MTJs.
Mechanical stretch was applied to the entire costal muscles of the left hemidiaphragm of the normal and mdx mice by passively stretching the muscle in the direction of the muscle fibers by applying passive tension of 
0.4 N/cm. This is equivalent to a passive stress of 11 N/cm2. Mechanical stretch of 50% from the unstressed state was achieved in the normal diaphragm. Unstressed length or length of excised muscle is the shortest length of the muscle; it is equivalent to the length of the muscle at a lung volume of total lung capacity. Optimal length is 125% of this unstressed length. Therefore, our stretch of 50% placed the diaphragm at a length that is equivalent to 120% of optimal muscle length. The abdominal wall was stretched along the fibers of the external oblique muscle, and biceps femoris was stretched along its muscle fibers. The magnitude of tension applied to both the abdominal wall and the biceps femoris was the same as that applied to the diaphragm muscle (0.4 N/cm).
In each of these protocols, the muscle was held in the stretched state for 15 min. In one protocol that was designed to investigate the kinetics of the activation of NF-B and IKK and the degradation of IB, the diaphragm muscle was held at a stretched state for time periods varying between 2 and 60 min. The total time postmortem for these protocols did not exceed 80 min.
Electrophoretic mobility shift assays (EMSAs)
To determine NF-kB activation, EMSAs were performed as described previously (22)
with some modification. At the end of each stretch protocol described previously, the muscles were immediately frozen with dry ice and suspended at 1 mg muscle weight per 18 µl of low salt lysis buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml aprotinin, 0.5 mg/ml benzamidine) followed by mechanical grinding. Cells in the lysis buffer were allowed to swell on ice for 5 min followed immediately by two cycles of freeze-thaw lysis. The tubes containing the lysed muscle cells were then vortexed vigorously for 10 s, and the lysate was centrifuged for 10 s at 14,000 rpm. The supernatant (cytoplasmic extracts) was removed and saved at -70°C for further biochemical analysis. The nuclear pellet was resuspended in 4 µl of ice-cold high-salt nuclear extraction buffer (20 mM HEPES [pH 7.9], 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml aprotinin, 0.5 mg/ml benzamidine) per mg of original muscle weight and was incubated on ice for 30 min with intermittent vortexing. Samples were centrifuged for 5 min at 4°C, and the supernatant (nuclear extract) was either used immediately or stored at –70°C. The protein content was measured by the method of BioRad (Hercules, CA) protein assay reagent. EMSAs were performed by incubating 14 µg of nuclear extract with 16 fmol of the 32P-end-labeled NF-B consensus oligonucleotide 5'-CGCTTGATGAGTCAGCCGGAA-3' (Promega, Madison, WI) (underline indicates the binding sites of NF-B) for 15 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 oligonucleotide on 7.5% native polyacrylamide gel, using buffer containing 50 mM Tris, 200 mM glycine (pH 8.5), and 1 mM EDTA. For the supershift assays, a 5% gel was used. The gel was dried, and the radioactive bands were visualized and quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), using ImageQuant software. The specificity of the NF-B band was confirmed by using a mutated NF-B probe, cold competition assays, and supershift assays, using antibodies against p65 and p50 proteins.
IKK assay
The IKK assay was performed by an established technique (23)
. In brief, the IKK complex were precipitated by treating 600 µg cytoplasmic extracts with 1 µg anti-IKKß antibody overnight at 4°C, followed by treatment with 40 µl protein A/G-Sepharose beads (Amersham Biosciences, Arlington Heights, IL). After 2 h, the beads were washed three times with lysis buffer and three times with the kinase assay buffer and then resuspended in 20 µl of kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl2, 2 mM DTT, 20 µCi -ATP, 10 µM unlabeled ATP, and 2 µg of substrate GST-IB (amino acid residues 1–54). After incubation at 30°C for 15 min, the reaction was terminated by boiling with 20 µl of 2x sodium dodecyl sulfate (SDS) sample buffer for 3 min. Finally, the protein was resolved on 10% polyacrylamide gel, the gel was dried, and the radioactive bands were visualized and quantitated by PhosphorImager, using ImageQuant Software (Molecular Dynamics, Sunnyvale, CA). Total amounts of IKKß in each sample were determined by Western blotting, using anti-IKKß.
Western blot
Fifty to seventy micrograms of cytoplasmic protein extracts prepared as described previously were resolved on 9% SDS-PAGE (PAGE) gel. The proteins were then electrotransferred to a nitrocellulose membrane blocked with 5% nonfat milk and probed with suitable antibodies (1:1500) for 4–5 h. The blot was washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h, and finally detected by chemiluminescence (ECL, Amersham Biosciences). The amount of protein in the blots was quantified using densitometer and ImageQuant software (Molecular Dynamics).
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from 20–30 mg of diaphragm muscle with TRIzol reagent (Invitrogen Life Technologies) following the manufacturer’s suggestions. Total RNA samples (2 µg) were reverse transcribed in 20 µl reaction volume using oligo-(dT)12–18 primers and SuperScript II RNase H– reverse transcriptase (Invitrogen Life Technologies) following the manufacturer’s instructions. Ten percent of the reverse transcribed reaction volume (2 µl, corresponding to 200 ng of original input RNA) was used for each subsequent 50 µl PCR reaction. To find out the number of PCR cycles in the exponential phase of amplification, a PCR master mix was prepared that contained 10 µl [-32P] dCTP in addition to the normal reaction components. The master mix was split into 10 aliquots, which were then subjected to PCR. Aliquots were removed from the thermal cycler at an interval of four cycles starting from cycle 16. The samples were resolved by electrophoresis on a polyacrylamide/urea gel. The gel was dried, and radioactive bands were visualized and quantitated by a PhosphorImager (Molecular Dynamics), using ImageQuant software. Cycle number was plotted against the log of the signal, and a straight line was obtained for samples in the exponential phase of amplification. Primer sequence used to amplify mouse TNF-, IL-1ß, and ß-globin are as follows: TNF-, 5'-GGC AGG TCT ACT TTG GAG TCA TTG C-3' (forward) and 5'-ACA TTC GAG GCT CCA GTG AAT TCG G-3' (reverse); IL-1ß, 5'-AAG GAG AAC CAA GCA ACG AC-3' (forward) and 5'-GAG ATT GAG CTG TCT GCT CA-3' (reverse); and ß-globin, 5'-CCT GCA GTG TCT GAT ATT GTT G-3' (forward) and 5'-AAC ACA CCA TTG CGA TGA A-3' (reverse).
The PCR reaction was carried out as follows: 3 min hot start at 94°C, followed by 30 cycles (1 min at 94°C, 30 s at 55°C, and 1 min at 72°C). The reaction was completed by a final amplification step at 72°C for 5 min. RT-PCR was also performed for ß-globin by using the primers described previously as a control to check the amount and integrity of the total RNA samples. The specificity of the amplified products was confirmed by the sizes of the products and also by performing PCR reactions in the absence of reverse transcribed products.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B by mechanical stretchB in the mouse diaphragm muscle. The left hemidiaphragm from adult mice (170 ± 12 days) was stretched along the direction of the muscle fibers, and the muscle was kept at the stretched state for different periods of times ranging from 5 to 60 min. We then prepared the nuclear extracts from the muscle cells and analyzed them by EMSA. The time-course analysis revealed that the NF-B/DNA-binding activity was increased as early as 5 min after the application of mechanical stretch, peaked at 30 min, and declined thereafter (Fig. 1
A). The specificity of mechanical stretch-induced NF-B/DNA complex was confirmed by demonstrating that binding was disrupted in the presence of a 100-fold excess of unlabeled NF-kB oligonucleotide (Fig. 1A
, lane 8), and the labeled oligonucleotide with a mutated NF-B binding site failed to bind the NF-B protein (Fig. 1A
, lane 9). Different combinations of Rel/NF-B proteins can constitute an active NF-B heterodimer that binds to specific sequences in DNA (31)
. To know the constituents of NF-B complex visualized by EMSA in mechanically stretched muscle cells, we incubated nuclear extracts from the stretched muscles with antibody to either p50 or p65 or a combination of both subunits and then conducted EMSA. Antibody to either p50 or p65 subunits of NF-B or a combination of both shifted the band to a higher molecular weight (Fig. 1B)
, suggesting that the mechanical stretch-activated complex consisted mainly of p50 and p65 proteins. Irrelevant antibodies such as anti-Raf1 and anti-JNK1 had no effect on the mobility of NF-B.
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The effect of mechanical stretch is not diaphragm muscle-specific because NF-B is also activated in abdominal and biceps femoris muscles in response to a mechanical stretch that is equivalent to that applied to the diaphragm (Fig. 1C)
. A quantative estimation of NF-B activation from six independent experiments of diaphragm muscle is presented in Fig. 1D
. These results suggest that mechanical stretch activates the NF-kB DNA binding activity in skeletal muscles.
Effect of mechanical stretch on the degradation of IB
The activation of NF-B by inflammatory cytokines such as TNF- is achieved through the phosphorylation of IB at ser-32 and ser-36 residue followed by its polyubiquitination and degradation (2)
. The degradation of IB leads to the nuclear translocation of NF-B. Whether mechanical stretch-induced activation of NF-B is associated with the degradation of IB is not clear. Muscles from the diaphragm were stretched by the application of mechanical stretch, and the muscle samples were held in the stretched state for different time periods. The cytoplasmic extracts from those mechanically stretched muscles were subjected to Western blot analysis, using IB-specific antibodies. The kinetics of IB protein showed a significant decrease in IB level after 15 min of applying the mechanical stretch (Fig. 2
). The effect of mechanical stretch on the degradation of IB was specific, because the application of mechanical stretch did not alter the level of an unrelated protein ß-actin during this time period (Fig. 2A
, lower panel).
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Effect of mechanical stretch on the activation of IKK
The degradation of IB is preceded by its phosphorylation by the IKK at ser-32 and ser-36 residue (24)
. We investigated the effects of mechanical stretch on the activation of IKK in the diaphragm. Muscles were mechanically stretched and held at the stretched state for different time intervals, and the activity of IKK in the stretched muscle was measured using GST-IB (1–54) protein. A significant increase in the IKK activity was observed as early as 5 min after the application of the mechanical stretch, which peaked at 15 min and decreased thereafter (Fig. 3
). The level of IKKß was unaffected by the application of mechanical stretch, suggesting that mechanical stretch only increases the activity of IKK without effecting the cellular level of IKK subunits.
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Role of Ca2+ influx in mechanical stretch-induced activation of NF-B
Previous studies have reported that mechanical stretch-dependent cellular responses are associated with an increase in the intracellular mobilization of Ca2+, mainly through the stretched activated (SA) channel and calcium channels (25
26
27)
. It remains unclear whether these pathways are involved in the activation of NF-B by mechanical forces in skeletal muscles. To investigate the role of Ca2+, the diaphragm muscles were pretreated for 30 min with either 25 µM gadolinium(III) chloride (a specific inhibitor of SA channels) or with 10 µM nifedipine (a classical inhibitor of Ca2+ channels) followed by stretching the muscle in the axial direction for 15 min. Pretreatment of cells with nifedipine did not have any effect on the activation of NF-B, whereas Gd3+ ions only marginally inhibited the activation of NF-B. Furthermore, although not statistically significant, the removal of Ca2+ from physiological solution seemed to marginally decrease the activation of NF-B (Fig. 4
). These results indicate that Ca2+ influx was apparently not necessary for the mechanical stretch-induced activation of NF-B in normal skeletal muscle.
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NAC inhibits the mechanical stretch-induced activation of NF-B
The role of oxygen free radicals as effectors in the signal transduction of TNF and other cytokines has been extensively described in the literature. Recent reports indicate that H2O2 plays an important role as a second messenger in signal transduction pathways that regulate the activity of NF-B (28
, 29)
. To determine whether mechanical stretch-induced NF-B/DNA binding activity involves reactive oxygen intermediates (ROI), we examined the effects of NAC, a free radical scavenger, on the activation of NF-B by mechanical stretch. Pretreatment of diaphragm muscles for 1 h with NAC (10–30 mM) inhibited the mechanical stretch-induced activation of NF-B in a dose-dependent manner, suggesting that in response to mechanical stretch, activation of NF-B involves ROI (Fig. 5
).
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Activation of NF-B in mdx mouse, a model for DMD
DMD, a severe muscle-wasting disease, is caused by the absence of the structural protein dystrophin, which provides mechanical reinforcement to the membranes of myocytes (13)
. Lack of dystrophin makes the skeletal muscles less compliant to stretches such as mechanical loading and osmotic shocks (16
17
18)
. The pathogenesis of DMD is frequently studied in the dystrophic mdx mouse model (30)
. Unlike hind limb muscles, the diaphragm muscle in the mdx mouse fails to regenerate (31)
. In mdx mice, muscular dystrophy starts to occur at the age of 3 wk and is characterized by recurrent signs of degeneration-regeneration of skeletal muscle (32
33
34)
. We studied the level of NF-B activity in diaphragm muscle from mdx mice at 15, 18, 30, and 60 days of age. Compared with controls, the NF-B activity was significantly higher in mdx mice, even in 15-day-old mice. The data demonstrate clearly that NF-B activation precedes the onset of muscular dystrophy (Fig. 6
A, upper panel, and Fig. 6B
). The increased activity of NF-B in mdx mice was specific to muscles, because there was no difference in the level of NF-B activity in liver between normal and mdx mice (Fig. 6A
, lower panel). The supershift analysis of the NF-B/DNA complex from mdx mice showed that the complex mainly contains p50 and p65 proteins (Fig. 6C)
. In addition, the level of IkB was also lower in the diaphragm muscle from mdx mice compared with the corresponding control mice (Fig. 6D)
. These results clearly show that skeletal muscle-specific activation of NF-B occurs in mdx mice even before the onset of muscular dystrophy.
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Expression of TNF- and IL-1ß in the diaphragm muscles of the mdx mice
The promoter region of either TNF- or IL-1ß contains the consensus B sequence (2)
. Therefore, we examined the expression of these cytokines in mdx and control mice of different ages. A significantly higher level of TNF- and IL-1ß was observed in mdx mice compared with controls (Fig. 7
). The kinetics of the expression of TNF- and IL-1ß shows that the level of these cytokines starts increasing even before the onset of muscle dystrophy. These results thus suggest that skeletal muscle-specific activation of NF-B in mdx mice may lead to an augmented level of TNF- and IL-1ß.
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Mechanical stretch has a synergistic effect on NF-B activation in mdx mice
The effect of mechanical stretch on the NF-B/DNA binding activity was also studied in mdx mice. The diaphragm muscle from 4-wk-old control as well as mdx mice was mechanically stretched in the direction of the muscle fibers, and muscles were kept in the stretched state for 15 min. We determined the level of NF-B activity by using EMSA. Interestingly, mechanical stretch applied to the diaphragm muscle led from the mdx mouse to a further augmentation in NF-B/DNA-binding activity (Fig. 8
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B in skeletal muscle fibers. Mechanical stretch of diaphragm muscle rapidly activates the NF-B, possibly through the generation of free radicals. Activation of NF-B in response to mechanical stretch is associated with an increase in IKK activity and a concomitant degradation of IB protein. Furthermore, the Ca2+ influx through either SA or Ca2+ channels is not required for mechanical stress-induced activation of NF-B in skeletal muscle. We also found that when compared with controls, the basal level of NF-B/DNA binding activity and the mRNA level of TNF- and IL-1ß are very high in the mdx mice. In addition, mechanical stretch of the diaphragm further augments the level of NF-B activity in the mdx mouse. Our experiments support the hypothesis that mechanical stretch activates NF-B in skeletal muscles, which might initiate the expression of several NF-kB-regulated inflammatory molecules. This in turn further augments the activity of NF-B in an autocrine fashion (Fig. 9
). The increased expression of these proinflammatory molecules might lead to muscular dystrophy.
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Muscle wasting is a general term that refers to the loss of skeletal muscles primarily through the ubiquitin-proteasome pathway (6)
. Muscle wasting is common in many diseases, including various muscular dystrophies (12)
, AIDS (35)
, cancer (36)
, and chronic heart failure (37)
. The abnormal increase in the level of proinflammatory molecules such as TNF- and IL-1ß has been suggested as the major mechanism that triggers events leading to muscle wasting (7
, 8
, 38
, 39)
. In vitro, incubation of the murine muscle cell line C2C12 as well as primary cultures from the rat skeletal muscles with TNF- resulted in a dose-dependent inhibition in the total protein concentration as well as the loss of adult myosin heavy chain (40
, 41)
. Many of the effects of TNF- and IL-1ß are attributed to their ability to activate the transcription factor NF-B. The inhibition of NF-B activity has been shown to provide a protective mechanism against TNF--induced protein degradation in C2C12 cells (42)
. Precisely how activation of NF-B leads to muscle loss is not clear. However, there is evidence in support of three possible mechanisms: 1) The activation of NF-B could enhance the activity of the ubiquitin/proteasome pathway, as has been previously suggested (6
, 43
, 44)
; 2) it could also lead to an augmented expression of several inflammatory molecules such as IL-1ß, IL-6, TNF-, cell adhesion molecules, and matrix metalloproteinase-9, which might directly or indirectly promote muscle wasting (2
, 6)
; and 3) it has been shown to interfere in the process of skeletal muscle differentiation, which is required for repair of damaged tissue (45
46
47)
.
Although, skeletal muscle wasting in response to inflammatory cytokines has been recently studied, not much work has been performed on the role of other mechanical factors, particularly cell stretching in the initiation of the process of muscle wasting. Skeletal muscles, like many other adherent cells, are routinely subjected to mechanical forces (48)
. Loading of skeletal muscle causes hypertrophy, whereas unloading leads to muscle atrophy (49)
. A recent and interesting study has reported the activation of an NF-B-dependent reporter gene in skeletal muscle during disuse atrophy in rats (11)
. This study showed that the nuclear levels of Bcl3, p50, and c-Rel proteins were increased by muscle unloading, and the NF-B/DNA complex contained mainly the Bcl3, p50and c-Rel proteins and not the other proteins of the NF-B family, especially p65, which is predominately present in the classical NF-B pathway. Based on these findings Hunter et al. (11)
proposed that unloading of skeletal muscles causes the activation of an alternative NF-B signaling pathway, which is commonly active during limb morphogenesis and insulin growth factor-induced muscle differentiation and involves the activation of the IKK catalytic subunit of the IKK complex (3
, 11)
.
We used an ex vivo system that is free of neuronal and hormonal effects to investigate the effect of mechanical stretch on the activation of NF-B in skeletal muscles. We used intact muscles from the left hemidiaphragm, which avoids the potential trauma that might be associated with muscle dissection. Our results demonstrate that DNA binding activity of NF-B is rapidly increased in response to mechanical stretch. The kinetics of the activation of NF-B and IKK suggests that NF-B activation is preceded by the activation of IKK and the subsequent degradation of IB protein. Although we used IKKß antibody to immunoprecipitate and subsequent kinase assay, it does not rule out possible activation of the IKK subunit in the IKK complex. However, because the activation of NF-B was associated with the degradation of IB, these results suggest that mechanical stretch activates NF-B via the canonical IB phosphorylation-degradation cycle pathway that requires activation of IKKß. This is further supported by the fact that use of both anti-p65 and anti-p50 antibodies supershifted the NF-B band in the mechanically stretched muscles to a higher molecular weight, showing that the active NF-B complex contains mainly p50 and p65 proteins (Fig. 1B)
. Taken together, the published report by Hunter et al. (11)
and the data that we provide in the present study suggest that both unloading and loading activate the NF-B pathways; unloading leads to the activation of the NF-B pathway that requires IKK activation, whereas loading activates the canonical NF-B pathway.
Activation of mechanosensitive ion channels has been proposed as the transduction mechanism between mechanical stretch and various cellular responses (50
, 51)
. Recently, Inoh et al. showed that direct Ca2+ influx through the SA channels is required for the cyclic stretch-activated NF-B in cultured human fibroblast cells (25)
. The level of intracellular Ca2+ in skeletal muscles has been reported to increase in response to mechanical forces, and this response is inhibited in the presence of Gd3+ ion or in Ca2+-depleted solution (50
51
52)
. An earlier report also suggests that elevation of intracellular Ca2+ concentration causes an increased elevation of ROI in mitochondria that leads to the translocation of NF-B from cytoplasm to nucleus (53)
. We investigated the role of free radicals and of Ca2+ influx in stress-induced activation of NF-B in skeletal muscles. Interestingly, only a marginal (not significant) effect on NF-B activation was observed when the diaphragm muscles were stretched in the presence of SA channel inhibitor or in Ca2+-free incubation medium (Fig. 4)
. Similarly, the inhibitors of Ca2+ channel did not affect the activation of NF-B in response to mechanical stretch. However, the activation of NF-B in skeletal muscles seems to involve the generation of ROI, because the treatment of muscle cells with NAC, a free radical scavenger (54)
, completely inhibited the stress-induced activation of NF-B (Fig. 5)
. The precise role of free radicals in the activation of NF-B in skeletal muscle in response to mechanical stretch is still enigmatic. However, several other studies suggest that free radicals act as a second messenger in the activation of NF-B in response to NF-B-activating stimuli such as PMA (phorbol 12 myristate 13-acetate) and TNF- (40
, 54)
.
Another important finding of the present investigation is the very high level of NF-B/DNA binding activity in the diaphragm of mdx mice, which was further enhanced by mechanical stretch. Muscle dystrophies are inherited myogenic disorders characterized by progressive muscle wasting (12)
. Although higher levels of TNF- and other inflammatory molecules have been reported in patients with DMD (7
8
9)
, the molecular mechanism that leads to elevated levels of these cytokines is not understood. Interestingly, we observed that NF-B activity was high in skeletal muscles of mdx mice even in the absence of applied mechanical stretch. In control mice, the activity of NF-B was high only at 15 days, and it decreased with age. Slightly higher NF-B activity in control mice at the age of 15 days could be attributed to muscle development in these immature mice. In contrast to this, NF-B activity was significantly higher in 15-day-old mdx mice compared with the corresponding control, which was not decreased with age (Fig. 6A
, upper panel, and Fig. 6B
). Furthermore, the supershift analysis of the NF-B/DNA complex and the degradation of IB protein suggest that the canonical NF-B pathway is predominately active in mdx mice (Fig. 6)
. We also found that the level of TNF- and IL-1ß increases in mdx mice even before the onset of muscular dystrophy (Fig. 8)
. Because both TNF- and IL-1ß contain consensus B sequence in their promoter region, the increased expression of these molecules in the skeletal muscles of mdx mice could be a direct result of the activation of NF-B.
Previously published work on the histological analysis of the diaphragm from mdx mice (32
33
34)
and our structural data (data not shown) demonstrate that muscular necrosis in these mice starts at 3 wk and peaks at 4 wk of age. It is therefore possible that activation of NF-B may lead to the accumulation of catabolic cytokines such as TNF-, and once these cytokines reach a certain threshold level, muscle injury and/or dystrophy may occur. Indeed, our results show that the level of TNF- is very high in 23-day-old mdx mice (Fig. 8)
. At that age skeletal muscle necrosis in the mdx mouse is significant and the activity of certain proteases is high (33)
.
The molecular events that lead to the activation of NF-B in mdx mice are not known. Lack of dystrophin in mdx causes skeletal muscles to be more susceptible to mechanical injury (16
17
18)
. It is therefore possible that mechanical stretch of dystrophin-deficient muscles of the mdx mouse, which occurs during normal activity, leads to an up-regulation of the NF-B activity. Based on the results presented here, we suggest that persistent stimulation of skeletal muscle fibers by mechanical forces or positive feedback loops that could involve proinflammatory cytokines such as TNF- and IL-1ß results in the over stimulation of NF-B and the development of muscular dystrophy (Fig. 9)
.
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ACKNOWLEDGMENTS |
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Received for publication July 9, 2002. Accepted for publication September 25, 2002.
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