Inflammatory Cytokine-Induced Muscle Atrophy and Weakness Can Be Ameliorated by an Inhibition of TGF-β-Activated Kinase-1

Abstract

Chronic inflammation causes muscle wasting. Because most inflammatory cytokine signals are mediated via TGF-β-activated kinase-1 (TAK1) activation, inflammatory cytokine-induced muscle wasting may be ameliorated by the inhibition of TAK1 activity. The present study was undertaken to clarify whether TAK1 inhibition can ameliorate inflammation-induced muscle wasting. SKG/Jcl mice as an autoimmune arthritis animal model were treated with a small amount of mannan as an adjuvant to enhance the production of TNF-α and IL-1β. The increase in these inflammatory cytokines caused a reduction in muscle mass and strength along with an induction of arthritis in SKG/Jcl mice. Those changes in muscle fibers were mediated via the phosphorylation of TAK1, which activated the downstream signaling cascade via NF-κB, p38 MAPK, and ERK pathways, resulting in an increase in myostatin expression. Myostatin then reduced the expression of muscle proteins not only via a reduction in MyoD1 expression but also via an enhancement of Atrogin-1 and Murf1 expression. TAK1 inhibitor, LL-Z1640-2, prevented all the cytokine-induced changes in muscle wasting. Thus, TAK1 inhibition can be a new therapeutic target of not only joint destruction but also muscle wasting induced by inflammatory cytokines.

Keywords: IL-1β; MyoD1; TAK1 inhibitor; TNF-α; myostatin.

Figure 1

LLZ alleviates inflammation and arthritis in SKG/jcl mice with mannan-induced inflammatory cytokine excess. (A) Western blot analysis of serum concentration of TNF-α and IL-1β. Serum levels were increased in SKG/Jcl mice treated with mannan. (B) Accumulated weekly food intakes are expressed as g/g body weight (BW) (n = 4 for each group). (C) Weekly change in body weight from 8 to 12 weeks of age (n = 12 for each group). Data are means ± SE for 12 mice, * p < 0.05 vs. control or control group with LLZ. (D) Change in arthritis score measured at all 4 paws in each mouse at 8, 10, and 12 weeks. The sum of scores for 4 paws in each mouse is presented. Data are means ± SD for 4 mice per each group, * p < 0.05, ## p < 0.001 vs. mannan group.

Figure 2

LLZ ameliorates inflammatory cytokine-induced muscle wasting. (A) Hindlimb muscle volume corrected by body weight. Data are expressed as means ± SE for 10 mice per each group, * p < 0.05. (B) Maximum grip strength corrected by body weight. Five consecutive measurements were recorded per mouse, and the average of the three best scores for each mouse is presented. Data are means ± SE for 12 mice per each group, * p < 0.05 vs. mannan + LLZ group.

Figure 3

Histological images of cross-sections of anterior tibialis and soleus muscles stained by NADH-TR. Granular muscle fibers represent type I slow-twitch fibers, and lighter staining fibers represent type II fast-twitch fibers. Scale bar = 50 μm.

Figure 4

LLZ ameliorates inflammatory cytokine-induced muscle atrophy. (A) Average myofiber area of type I slow-twitch and type II fast-twitch fibers and (B) average myofiber number in anterior tibialis and soleus muscle. Data are expressed as means ± SE for 30 measurements per a mouse from 3 mice, * p < 0.05, ** p < 0.01, # p < 0.005. Gray bars represent type I slow-twitch fibers and white bars represent fast-twitch fibers.

Figure 5

Enhancement of myostatin expression and suppression of MyoD1 expression by TNF-α were reversed by LLZ at 6 μM or higher in C2C12 cells. Relative mRNA levels by qPCR of (A) myostatin and (B) MyoD1 in C2C12 cells. Data are expressed as means ± SD for 5 measurements, * p < 0.05, ** p < 0.01, # p < 0.005, ## p < 0.001.

Figure 6

Enhancement of myostatin expression and suppression of MyoD1 expression by TNF-α and IL-1β were reversed by LLZ in C2C12 cells. Relative mRNA expression levels by qPCR of (A) myostatin and (B) MyoD1 in C2C12 cells. Data are expressed as means ± SD for 5 measurements, * p < 0.05, ** p < 0.01, # p < 0.005, ## p < 0.001.

Figure 7

Effects of TNF-α and IL-1β on the mRNA expression of muscle-specific proteins were reversed by LLZ in C2C12 cells. Relative mRNA levels by qPCR of muscle-specific fast-twitch proteins, (A) Myh1, (B) Myh4, and a slow-twitch protein, (C) Myh7, in C2C12 cells. Data are expressed as means ± SD for 5 measurements, * p < 0.05, ** p < 0.01, # p < 0.005, ## p < 0.001.

Figure 8

Effects of TNF-α and IL-1β on the mRNA expression of muscle atrophic factors and myotube size were reversed by LLZ in C2C12 cells. Relative mRNA levels by qPCR of (A) Atrogin-1 and (B) Murf-1 in C2C12 cells. Data are expressed as means ± SD for 5 experiments. (C) Size of myotube differentiated from C2C12 cells. Data are means ± SE for 10 measurements per each culture dish from 5 each treatment group, * p < 0.05, # p < 0.005, ## p < 0.001.

Figure 9

TNF-α and IL-1β enhanced TAK1 signaling, and LLZ abrogated TAK1 signaling in C2C12 cells. Western blot analysis of intracellular IκBα, phosphorylated TAK1, p38, and ERK in C2C12 cells treated with (A) TNF-α or (B) IL-1β in the presence or absence of LLZ.