Cathepsin K activity controls cachexia‐induced muscle atrophy via the modulation of IRS1 ubiquitination

Abstract

Background Cachexia is a complicated metabolic disorder that is characterize by progressive atrophy of skeletal muscle. Cathepsin K (CTSK) is a widely expressed cysteine protease that has garnered attention because of its enzymatic and non‐enzymatic functions in signalling in various pathological conditions. Here, we examined whether CTSK participates in cancer‐induced skeletal muscle loss and dysfunction, focusing on protein metabolic imbalance. Methods Male 9‐week‐old wild‐type (CTSK+/+, n = 10) and CTSK‐knockout (CTSK−/−, n = 10) mice were injected subcutaneously with Lewis lung carcinoma cells (LLC; 5 × 105) or saline, respectively. The mice were then subjected to muscle mass and muscle function measurements. HE staining, immunostaining, quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and western blotting were used to explore the CTSK expression and IRS1/Akt pathway in the gastrocnemius muscle at various time points. In vitro measurements included CTSK expression, IRS1/Akt pathway‐related target molecule expressions, and the diameter of C2C12 myotubes with or without LLC‐conditioned medium (LCM). An IRS1 ubiquitin assay, and truncation, co‐immunoprecipitation, and co‐localization experiments were also performed. Results CTSK+/+ cachectic animals exhibited loss of skeletal muscle mass (muscle weight loss of 15%, n = 10, P < 0.01), muscle dysfunction (grip strength loss > 15%, n = 10, P < 0.01), and fibre area (average area reduction > 30%, n = 5, P < 0.01). Compared with that of non‐cachectic CTSK+/+ mice, the skeletal muscle of cachectic CTSK+/+ mice exhibited greater degradation of insulin receptor substrate 1 (IRS1, P < 0.01). In this setting, cachectic muscles exhibited decreases in the phosphorylation levels of protein kinase B (Akt308, P < 0.01; Akt473, P < 0.05) and anabolic‐related proteins (the mammalian target of rapamycin, P < 0.01) and increased levels of catabolism‐related proteins (muscle RING‐finger protein‐1, P < 0.01; MAFbx1, P < 0.01) in CTSK+/+ mice (n = 3). Although there was no difference in LLC tumour growth (n = 10, P = 0.44), CTSK deletion mitigated the IRS1 degradation, loss of the skeletal muscle mass (n = 10, P < 0.01), and dysfunction (n = 10, P < 0.01). In vitro, CTSK silencing prevented the IRS1 ubiquitination and loss of the myotube myosin heavy chain content (P < 0.01) induced by LCM, and these changes were accelerated by CTSK overexpression even without LCM. Immunoprecipitation showed that CTSK selectively acted on IRS1 in the region of amino acids 268 to 574. The results of co‐transfection of IRS1‐N‐FLAG or IRS1‐C‐FLAG with CTSK suggested that CTSK selectively cleaves IRS1 and causes ubiquitination‐related degradation of IRS1. Conclusions These results demonstrate that CTSK plays a novel role in IRS1 ubiquitination in LLC‐induced muscle wasting, and suggest that CTSK could be an effective therapeutic target for cancer‐related cachexia.

Cathepsin S deficiency improves muscle mass loss and dysfunction via the modulation of protein metabolism in mice under pathological stress conditions

“…In this study, we found that stress causes an increase in white blood cells, and increases in the levels of pro‐inflammatory cytokines in the blood (IFN‐γ, IL‐1ɑ, IL‐1β, IL‐10, IL‐18) and in the skeletal muscles (MCP‐1, TNF‐ɑ, ICAM‐1, VCAM‐1, IL‐17, IL‐18). Because inflammatory cytokines have been shown to affect CTSS expression, 2,26 we proposed that up‐regulation of CTSS by the inflammatory stimulators functions as an important mediator of stress‐related muscle disorder in mice.…”

“…These pathological disease states cause skeletal muscle atrophy by imbalanced protein synthesis and degradation in skeletal muscle 2,3 . Skeletal muscle atrophy is often accompanied by an elevation in inflammatory cytokines and oxidative stress production, which induce harmful changes in the metabolism of proteins, carbohydrates, and lipids 2,4 . Emerging evidence suggests that biological oxidative and inflammatory stimulators increase the expression of several proteases, including members of the matrix metalloproteinases and cysteinyl cathepsins (i.e., CTSS and CTSK), which then modulate resident cell events (migration, invasion, apoptosis, and proliferation) and extracellular matrix remodeling in inflammatory and metabolic disorders 2,5,6,7,8,9 .…”

“…2,3 Skeletal muscle atrophy is often accompanied by an elevation in inflammatory cytokines and oxidative stress production, which induce harmful changes in the metabolism of proteins, carbohydrates, and lipids. 2,4 Emerging evidence suggests that biological oxidative and inflammatory stimulators increase the expression of several proteases, including members of the matrix metalloproteinases and cysteinyl cathepsins (i.e., CTSS and CTSK), which then modulate resident cell events (migration, invasion, apoptosis, and proliferation) and extracellular matrix remodeling in inflammatory and metabolic disorders. 2,5,6,7,8,9 Although it is well-known that chronic stress causes inflammatory actions and oxidative stress production to promote cardiovascular disease initiation and progression in humans and animals under various pathological conditions, [5][6][7] it remains largely uncertain CPS causes skeletal muscle atrophy and dysfunction.…”

Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters

“…We also observed that tdT was expressed not only in skeletal muscle (muscle fibers and satellites) but also in smooth muscle in the artery, indicating a role of Ctsk in muscles and blood vessels; an increased serum level of CTSK was also observed in patients with coronary artery disease ( 50 ) and is involved in muscle degeneration and regeneration. ( 51 ) In addition, we defined two types of neural cells expressing tdT: pyramidal cells in the hippocampus and Purkinje neurons in the cerebellum. tdT expression in the brain also implies its multiple roles in the CNS.…”

Eldecalcitol prevents muscle loss by suppressing PI3K/AKT/FOXOs pathway in orchiectomized mice

“…An increase in AKT level during hypertrophy and a decrease in AKT level during atrophy, has been demonstrated in mice ( Szewczyk et al, 2007 ). Besides, AKT has been found to produce a marked effect in muscle atrophy caused by a variety of pathogeny including diabetes type II-related sarcopenia, muscle atrophy induced by cachexia and glucocorticoid ( Cetrone et al, 2014 ; Meng et al, 2022 ; Mishra et al, 2022 ). In addition, AKT involves in the regulation of different effectors, including ion channels that regulate cell proliferation in cell lines and skeletal muscle ( Cetrone et al, 2014 ; Maqoud et al, 2018 ).…”