Skeletal muscle glycoprotein 130's role in Lewis lung carcinoma-induced cachexia

Abstract

Chronic inflammation is associated with cachexia-induced skeletal muscle mass loss in cancer. Levels of IL-6 cytokine family members are increased during cancer-related cachexia and induce intracellular signaling through glycoprotein130 (gp130). Although muscle STAT3 and circulating IL-6 are implicated in cancer-induced muscle wasting, there is limited understanding of muscle gp130's role in this process. Therefore, we investigated the role of skeletal muscle gp130 (skm-gp130) in cancer-induced alterations in the regulation of muscle protein turnover. Lewis lung carcinoma (LLC) cells were injected into 8-wk-old skm-gp130-knockout (KO) mice or wild-type mice. Skeletal muscle loss was attenuated by 16% in gp130-KO mice, which coincided with attenuated LLC-induced phosphorylation of muscle STAT3, p38, and FOXO3. gp130 KO did not restore mTOR inhibition or alter AMP-activated protein kinase (AMPK) expression. The induction of atrogin expression and p38 phosphorylation in C2C12 myotubes exposed to LLC-treated medium was attenuated by gp130 inhibition, but mTOR inhibition was not restored. STAT signaling inhibition in LLC-treated myotubes did not attenuate the induction of p38 or AMPK phosphorylation. We concluded that, during LLC-induced cachexia, skm-gp130 regulates muscle mass signaling through STAT3 and p38 for the activation of FOXO3 and atrogin, but does not directly regulate the suppression of mTOR.

Keywords: C2C12 myotube; IL-6; STAT3; cancer; inflammation; protein turnover.

Figure 1.

Mouse tissue expression of gp130. A) gp130 was differentially expressed in tissues. B) Differential expression of gp130 mRNA in the soleus (Sol), gastrocnemius (Gastroc), and TA muscles of C57BL/6 mice. C) PCR analysis of gp130 mRNA (643 bp product size) in the gastrocnemius (Gas), soleus (Sol), and TA muscles and the liver, kidney, and heart of skm-gp130^+/+, skm-gp130^+/−, and skm-gp130^−/− mice. D) STAT3 protein phosphorylation in heterozygous and homozygous skm-gp130-KO mice after 2 wk of IL-6 overexpression. All values are means ± sem. *P < 0.05 vs. genotype vector control; ^†P < 0.05 vs. all other groups.

Figure 2.

Effect of skm-gp130 on development of cancer-induced cachexia. LLC tumor cells were implanted in 8-wk-old wild-type BL/6 and skm-gp130^−/− (skm-gp130) mice. Tumors were allowed to grow until 13 wk of age. A) Skm-gp130 expression changed with LLC-induced cachexia in wild-type and skm-gp130 mice in the gastrocnemius. ND, not detected. B) Tumor mass was measured at the time of death. C, D) Percentage change in body weight (BW) from genotype control (C) and percentage change in gastrocnemius muscle mass (D) were calculated from weights collected at the time of death. All values are means ± sem. ***P < 0.05 vs. BL/6 group.

Figure 3.

Effect of skm-gp130 on LLC-induced signaling. A) Western blot analysis of p-STAT3, total STAT3, p-AMPK, total AMPK, p-p38, total p38, p-NF-κB (p-p65), and total NF-κB protein expression. Ponceau staining verified equal loading. B) Western blot analysis of p-Akt (T308), p-Akt(S473), and total Akt. C) Western blot analysis of the mTOR signaling proteins p-4EBP1, total 4EPB1, p-S6, total S6, p-FOXO3, total FOXO3, and atrogin. Ponceau staining verified equal loading. B, C) Graphs represent the ratio of phosphorylated to total protein levels. D) Levels of ubiquitinated proteins were quantified by Western blot analysis, with equal loading verified by Ponceau stain. Dashed line indicates different sections of the same gel. E) Skeletal muscle mRNA expression of IGF-1, REDD1, and IL-6 was measured in the gastrocnemius. All values are means ± se. Two-way ANOVA was used to analyze the effect of skm-gp130 and LLC. *P < 0.05 vs. BL/6 control; **P < 0.05 vs. BL/6 LLC; ^†P < 0.05 vs. all other groups.

Figure 4.

Effect of LCM on myosin heavy chain level, STAT3 signaling, and protein turnover regulation in C2C12 myotubes. A) Myotube atrophy induced by 72 h LCM exposure was measured with protein levels of myosin heavy chain. B) Inflammatory signaling of STAT3 induced by administration of LCM for 4 or 72 h. C) Protein expression of p-Akt, total Akt, p-AMPK, total AMPK, p-mTOR, total mTOR, p-S6RP, total S6RP, p-4EBP1, and total 4EPB1 after 72 h treatment with LCM was measured by Western blot analysis. D) Protein expression of p-FOXO3, total FOXO3, atrogin, and GAPDH with various LLC levels was measured by Western blot analysis. Graphs represent the ratio of phosphorylated to total protein levels. Values are means ± sem. Data were analyzed with 1-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005 vs. control group.

Figure 5.

Effect of IL-6 inhibition on LLC-induced signaling. A) p-STAT3 and total STAT3 protein expression was measured via Western blot analysis in fully differentiated C2C12 cells treated with IL-6 (100 ng/ml) or LCM (25%) for 4 h, with or without IL-6r Ab (1:1000). B) Protein synthesis was measured by the incorporation of puromycin into proteins in C2C12 cells treated for 72 h with LCM (25%), with or without IL-6r Ab (1:1000). Loading was verified with Ponceau staining. C) Protein expression of p-AMPK, total AMPK, p-Akt, total Akt, p-4EBP1, total 4EBP1, p-FOXO3, total FOXO3, atrogin, and GAPDH was measured by Western blot in C2C12 cells treated with LCM (25%) for 72 h, with or without IL-6r Ab (1:1000). Graph represents the ratio of phosphorylated to total protein levels. D) IL-6r Ab was administered acutely in vivo in mice with LLC tumors for 1 wk after tumor development. Western blot analysis measured LLC-associated signaling of STAT3, p38, Akt, S6RP, and atrogin-1. E) Protein synthesis was determined by Western blot 30 min after injection of puromycin, with loading verified by Ponceau staining. Dashed line indicates different sections of the same gel. Values are means ± se. Cell culture data were analyzed with 1-way ANOVA and in vivo data with the t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005 vs. control group.

Figure 6.

Effect of gp130/STAT signaling inhibition on LLC-induced signaling. Fully differentiated C2C12 cells were treated with LCM, with or without PDTC (a STAT/NF-kB inhibitor, 50 μM), LLL12 (a STAT3 specific inhibitor, 100 nM), and gp130r Ab (1:1000) for 4 h (A) or 72 h (B–F). A) p-STAT3 and total STAT3 protein expression was measured by Western blot analysis after 4 h treatment. B) Diameter of C2C12 myotubes after 72 h of LLC, PDTC, LLL12, or gp130r Ab administration. C) Relative protein synthesis rates were measured by Western blot after the incorporation of puromycin into proteins after 72 h of exposure to PDTC, LLL12, or gp130 Ab. Loading was verified by Ponceau staining. D–F) C2C12 signaling protein was measured by Western blot analysis after 72 h of exposure to gp130r Ab (D), LLL12 (E), or PDTC (F) in LCM. G) PDTC was administered for 1 wk to mice bearing LLC tumors. LLC-associated signaling of STAT3, P65, AMPK, P38, and Akt, was measured by Western blot analysis. Graphs represent ratio of phosphorylated to total protein levels. Values are means ± sem. Cell culture data were analyzed with 1-way ANOVA, and in vivo data were analyzed with the t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005 vs. control group; ^#P ≤ 0.05 vs. LLC group.