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 lipopolysac‐charide (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 lacta‐cystin 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 ubiq‐uitin‐proteasome and the autophagy‐lysosomal pathways.—Doyle, A., Zhang, G., Abdel Fattah, E. A., Eissa, N. T., Li, Y.‐P. Toll‐like receptor 4 mediates lipopolysac‐charide‐induced muscle catabolism via coordinate activation of ubiquitin‐proteasome and autophagy‐lysosome pathways. FASEB J. 25, 99–110 (2011). www.fasebj.org

Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor’s capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.

Upregulation of ubiquitin ligase atrogin1/MAFbx and muscle wasting are hallmarks of cancer cachexia; however, the underlying mechanism is undefined. Here, we describe a novel signalling pathway through which Lewis lung carcinoma (LLC) induces atrogin1/MAFbx upregulation and muscle wasting. C2C12 myotubes treated with LLC‐conditioned medium (LCM) rapidly activates p38 MAPK and AKT while inactivating FoxO1/3, resulting in atrogin1/MAFbx upregulation, myosin heavy chain loss, and myotube atrophy. The p38α/β MAPK inhibitor SB202190 blocks the catabolic effects. Upon activation, p38 associates with C/EBPβ resulting in its phosphorylation and binding to a C/EBPβ‐responsive cis ‐element in the atrogin1/MAFbx gene promoter. The promoter activity is stimulated by LCM via p38β‐mediated activation of the C/EBPβ‐responsive cis ‐element, independent of the adjacent FoxO1/3‐responsive cis ‐elements in the promoter. In addition, p38 activation is observed in the muscle of LLC tumour‐bearing mice, and SB202190 administration blocks atrogin1/MAFbx upregulation and muscle protein loss. Furthermore, C/EBPβ−/− mice are resistant to LLC tumour‐induced atrogin1/MAFbx upregulation and muscle wasting. Therefore, activation of the p38β MAPK–C/EBPβ signalling pathway appears a key component of the pathogenesis of LLC tumour‐induced cachexia.

Background: Activation of type IIB activin receptor (ActRIIB) in skeletal muscle leads to muscle atrophy because of increased muscle protein degradation. However, the intracellular signalling mechanism that mediates ActRIIB‐activated muscle catabolism is poorly defined.Methods: We investigated the role of p38β mitogen‐activated protein kinases (MAPK) in mediating ActRIIB ligand activin A‐activated muscle catabolic pathways in C2C12 myotubes and in mice with perturbation of this kinase pharmacologically and genetically.Results: Treatment of C2C12 myotubes with activin A or myostatin rapidly activated p38 MAPK and its effector C/EBPβ within 1 h. Paradoxically, Akt was activated at the same time through a p38 MAPK‐independent mechanism. These events were followed by up‐regulation of ubiquitin ligases atrogin1 (MAFbx) and UBR2 (E3α‐II), as well as increase in LC3‐II, a marker of autophagosome formation, leading to myofibrillar protein loss and myotube atrophy. The catabolic effects of activin A were abolished by p38α/β MAPK inhibitor SB202190. Using small interfering RNA‐mediated gene knockdown, we found that the catabolic activity of activin A was dependent on p38β MAPK specifically. Importantly, systemic administration of activin A to mice similarly activated the catabolic pathways in vivo, and this effect was blocked by SB202190. Further, activin A failed to activate the catabolic pathways in mice with muscle‐specific knockout of p38β MAPK. Interestingly, activin A up‐regulated MuRF1 in a p38 MAPK‐independent manner, and MuRF1 did not appear responsible for activin A‐induced myosin heavy chain loss and muscle atrophy.Conclusions: ActRIIB‐mediated activation of muscle catabolism is dependent on p38β MAPK‐activated signalling.

Cancer-induced cachexia, characterized by muscle wasting, is a lethal metabolic syndrome with undefined etiology. Current consensus is that multiple factors contribute to cancer-induced muscle wasting, and therefore therapy requires combinational strategies. Here, we show that Toll-like receptor 4 (TLR4) mediates cancer-induced muscle wasting by directly activating muscle catabolism as well as stimulating an innate immune response in mice bearing Lewis lung carcinoma (LLC), and targeting TLR4 alone effectively abrogate muscle wasting. Utilizing specific siRNAs we observed that LLC cell-conditioned medium (LCM)-treated C2C12 myotubes underwent a rapid catabolic response in a TLR4-dependent manner, including activation of the p38 MAPK−C/EBPβ signaling pathway as well as the ubiquitin-proteasome and autophagy-lysosome pathways, resulting in myotube atrophy. Utilizing a reporter cell-line it was confirmed that LCM activated TLR4. These results suggest that LLC-released cachexins directly activate muscle catabolism via activating TLR4 on muscle cells independent of immune responses. Critically, LLC tumor-bearing TLR4^−/−mice were spared from muscle wasting due to a blockade in muscle catabolic pathways. Further, tumor-induced elevation of circulating TNFα and interleukin-6 (IL-6) was abolished in TLR4^−/−mice. These data suggest that TLR4 is a central mediator and therapeutic target of cancer-induced muscle wasting.