Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways

Abstract

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.

Figure 1.

LPS induces myotube atrophy. A) LPS induces myotube MHC loss. C2C12 myotubes were treated with saline or 10 to 1000 ng/ml LPS for 48 h. MHC content in cell lysate was evaluated by Western blot analysis. B) LPS reduces myotube size. C2C12 myotubes were treated with saline or 100 ng/ml LPS for 48 h. Immunofluorescence staining with anti-MHC antibody revealed a distinct reduction in myotube diameter in LPS-treated myotubes. Scale bars = 50 μm. Myotube diameter was measured to evaluate myotube atrophy. *P < 0.05 vs. saline control; Student's t test.

Figure 2.

LPS-induced myotube atrophy is mediated by p38 MAPK. A) LPS activates AKT and p38 MAPK in myotubes. C2C12 myotubes were treated with 100 ng/ml LPS for indicated periods. Cell lysate was analyzed for phosphorylation of p38 MAPK (T180/Y182), AKT (S473), and FoxO 1/3a (FoxO1 T24/FoxO3a T32) by Western blot analysis. *P < 0.05 vs. 0 min; ANOVA. B) LPS-induced myotube atrophy is dependent on p38 MAPK activation. Myotubes were pretreated with SB202190 (10 μM) for 30 min prior to LPS treatment. In 48 h of LPS treatment, immunofluorescence staining of MHC was performed as described in Fig. 1, and myotube diameter was measured to evaluate myotube atrophy. *P < 0.05 vs. saline control; ^†P < 0.05 vs. LPS treatment; ANOVA. C) Activation of p38 MAPK by MKK6 induces MHC loss. Transduction of adenovirus encoding MKK6bE or GFP was initiated in C2C12 cells that had been differentiated for 48 h. In 48 h of transduction when myotubes formed, phospho-p38, p38, and MHC content in cell lysate was evaluated by Western blot analysis. *P < 0.05 vs. GFP-expressing myotubes; ANOVA. D) Activation of p38 MAPK by MKK6 induces myotube atrophy. Transduction of adenovirus encoding MKK6bE or GFP was performed as described in panel C with or without the presence of SB202190 (10 μM). In 48 h of transduction, immunofluorescence staining of MHC was performed and myotube diameter was measured to evaluate myotube atrophy. *P < 0.05 vs. GFP-expressing myotubes; ANOVA.

Figure 3.

LPS up-regulates atrogin-1/MAFbx expression and autophagosome formation in myotubes via the activation of p38 MAPK. A) LPS up-regulates the expression of atrogin-1/MAFbx and MuRF1 in myotubes. C2C12 myotubes were treated with saline or 100 ng/ml LPS for indicated periods. Real-time PCR was performed to determine the mRNA levels of the ubiquitin ligases in total cellular RNA that had been reverse transcribed into cDNA. *P < 0.05 vs. control; ANOVA. B) LPS up-regulation of atrogin-1/MAFbx is dependent on p38 MAPK activation. C2C12 myotubes were pretreated with 10 μM SB202190 prior to 2 h LPS treatment. Atrogin-1/MAFbx mRNA level was determined by real-time PCR. *P < 0.05 vs. control; Student's t test. Western blot analysis was performed to examine atrogin-1/MAFbx levels in C2C12 myotubes treated with LPS and/or SB202190 for 6 h. C) Activation of p38 MAPK by overexpressed active MKK6 up-regulates atrogin-1/MAFbx. Transduction of adenovirus encoding MKK6bE or GFP was initiated in C2C12 cells that had been differentiated for 48 h with or without the presence of 10 μM of SB202190. In 48 h of transduction, mRNA levels of atrogin-1/MAFbx and MuRF1 were determined by real-time PCR. *P < 0.05 vs. GFP; ^†P < 0.05 vs. MKK6bE; ANOVA. D) LPS induces LC3-II increase in myotubes. C2C12 myotubes were treated with saline or 100 ng/ml LPS for indicated periods. Myotubes was also treated with lysosome inhibitor chloroquine (CQ, 50 μM) for 24 h to induce LC3-II accumulation. An LC3B antibody that detects both LC3-I and LC3-II (Cell Signaling; 2775S) was used in Western blot analysis. E) LPS induces LC3-II increase in a p38-dependent manner. C2C12 myotubes were treated with saline or 100 ng/ml LPS for 8 h with or without pretreatment of 10 μM of SB202190. Myotubes were also treated with chloroquine for 8 h to induce LC3-II accumulation. An LC3B antibody that detects both LC3-I and LC3-II (Cell Signaling; 2775S) was used in Western blot analysis. LC3-II levels were normalized to the loading control β-actin. *P < 0.05 vs. saline control; ^†P < 0.05 vs. LPS treatment base; ANOVA. F) LPS up-regulates autophagosome formation in a p38-dependent manner. C2C12 myoblasts were transfected with a plasmid encoding GFP-LC3 and allowed to differentiate into myotubes. Myotubes were then treated with saline or 100 ng/ml LPS for 12 h with or without pretreatment with 10 μM of SB202190. Localization of expressed GFR-LC3 was examined under a fluorescence microscope. Scale bars = 100 μm. G) Activation of p38 MAPK by overexpressed active MKK6 up-regulates autophagosome formation. Adenovirus encoding MKK6bE or GFP was transduced into C2C12 cells as described above. LC3-II levels in myotubes were evaluated by Western blot analysis with an antibody that detects LC3-II but not LC3-I (Cell Signaling; 3866S). *P < 0.05 vs. saline control; ANOVA.

Figure 4.

TLR4 mediates LPS activation of p38 MAPK, UPP, ALP, and myotube atrophy. C2C12 myoblasts were transfected with TLR4-specific siRNA or control siRNA. After differentiation, myotubes were treated with 100 ng/ml of LPS or saline for various periods for the following analyses. A) TLR4 mediates LPS activation of p38 MAPK and AKT. After 30 min of LPS treatment, TLR4 levels and phosphorylation of p38 MAPK and AKT in myotubes were evaluated by Western blot analysis. B) TLR4 mediates LPS up-regulation of atrogin-1/MAFbx and MuRF1. Real-time PCR was performed to determine atrogin-1/MAFbx mRNA in myotubes treated with LPS for 2 h, and MuRF1 mRNA in myotubes treated with LPS for 3 h. *P < 0.05 vs. control siRNA; Student's t test. C) TLR4 mediates LPS stimulation of LC3-II increase. LC3-II levels were evaluated in myotubes treated with LPS for 8 h by Western blot analysis with an antibody that detects LC3-II but not LC3-I (Cell Signaling; 3866S). D) TLR4 mediates LPS-induced myotube atrophy. Myotubes treated with LPS for 48 h were subjected to immunofluorescence staining with an antibody against MHC. Myotube diameter was measured. *P < 0.05 vs. saline; Student's t test.

Figure 5.

LPS induces muscle atrophy in mice via the activation of the ALP, as well as the UPP. A) LPS administration up-regulates autophagosome formation in mouse TA. LPS (1 mg/kg) or saline was injected (i.p.) to male GFP-LC3 transgenic mice (8 wk of age). In 16 h, TA of the mice was collected, and cryosections were prepared for examination of GFP-LC3 localization using a fluorescence microscope. Scale bars = 50 μm. B) LPS-induced muscle proteolysis is partially mediated by the UPP. LPS (1 mg/kg) or saline was injected (i.p.) to wild-type male mice (8 wk of age). In 18 h, EDL was collected and tyrosine release was determined with or without the presence of 10 μM of lactacystin in incubation buffer. *P < 0.05 vs. saline control; ^†P < 0.05 vs. LPS control; ANOVA. C) Autophagy inhibitor 3-MA abolishes LPS-induced muscle proteolysis. Wild-type mice were pretreated with autophagy inhibitor 3-MA (10 mg/kg) or saline injection (i.p.) 1×/d for 4 d prior to LPS injection. In 18 h after LPS injection, EDL was collected, and tyrosine release was determined. *P < 0.05 vs. saline control; ^†P < 0.05 vs. LPS control; ANOVA. D) Autophagy inhibitor 3-MA blocks LPS up-regulation of autophagosome formation. Wild-type mice were pretreated with autophagy inhibitor 3-MA (10 mg/kg) or saline injection (i.p.) prior to LPS injection as described above. Lysate of TA collected in 16 h was subjected to Western blot analysis with an antibody that detects LC3-II but not LC3-I (Cell Signaling; 3866S). *P < 0.05 vs. saline control; ^†P < 0.05 vs. LPS treatment; ANOVA.

Figure 6.

TLR4 mediates LPS-induced muscle atrophy in mice by activating the UPP and the ALP. LPS (1 mg/kg) or saline was injected (i.p.) to TLR4^−/− or wild-type (WT) male mice (8 wk of age). TA and EDL were collected at 18 h after injection for analyses described below. A) TLR4 deficiency abolishes LPS-induced tyrosine release. Tyrosine release from EDL was determined. *P < 0.05 vs. saline control. B) TLR4 deficiency abolishes LPS up-regulation of atrogin-1/MAFbx and MuRF1 mRNA. Levels of mRNA of atrogin-1/MAFbx and MuRF1 in TA were determined by real-time PCR. *P < 0.05 vs. WT; Student's t test. C) TLR4 deficiency abolishes LPS up-regulation of atrogin-1/MAFbx and MuRF1 protein. Western blot analysis was performed to examine the protein level of atrogin-1/MAFbx and MuRF1 in the homogenate of TA. Protein bands were quantified by densitometry. *P < 0.05 vs. WT saline control; ANOVA. D) TLR4 deficiency abolishes LPS-induced LC3-II increase. LC3-II levels in TA were determined by Western blot analysis with an antibody that detects LC3-I, as well as LC3-II. *P < 0.05 vs. WT; Student's t test.

Figure 7.

p38 MAPK mediates TLR4 activation of the UPP and the ALP in mouse muscle. Wild-type or GFP-LC3 transgenic male mice (8 wk of age) were pretreated with daily injection (i.p.) of SB202190 (5 mg/kg) or vehicle for 4 d, followed by LPS (1 mg/kg) or saline injection (i.p.). TA and EDL were collected at indicated times for analyses described below. A) p38 MAPK mediates TLR4 up-regulation of autophagosome formation. Localization of GFP-LC3 in TA of GFP-LC3 transgenic mice was examined using a fluorescence microscope at 16 h after LPS injection. Scale bars = 50 μm. B) p38 MAPK mediates TLR4 up-regulation of LC3-II levels. LC3-II levels in wild-type TA collected at 18 h after LPS injection were determined using Western blot analysis with an antibody that detects LC3-II but not LC3-I (Cell Signaling; 3866S). *P < 0.05 vs. control; ANOVA. C) p38 MAPK mediates TLR4 up-regulation of atrogin-1/MAFbx. mRNA levels of atrogin-1/MAFbx and MuRF1 in wild-type TA collected 12 h after LPS injection were determined by real-time PCR. *P < 0.05 vs. LPS; Student's t test. D) p38 MAPK mediates TLR4 stimulation of tyrosine release. Tyrosine release from wild-type EDL collected at 18 h after LPS injection was determined. *P < 0.05 vs. control; ANOVA.