STAT3-induced myotube atrophy is FOXO1 dependent.
It has been reported that during muscle atrophy, ubiquitin-proteasome pathway activation and the expression of MuRF-1/Atrogin-1 are regulated by the transcription factor FOXO1 (Lokireddy et al., 2011; Stitt et al., 2004). Therefore, we hypothesized that STAT3 might regulate FOXO1 expression and activation in cachectic skeletal muscle. As shown in Figure 6a, the FOXO1 expression level was significantly increased in C26 cachectic mice muscle, which was down-regulated upon in vivo17DMAG treatment (Figure 6a upper part, Figure S4b), and a similar result was observed in C2C12 myotubes when it was subjected to C26 CM treatment (Figure 6b lower part). The expression of FOXO1 in C26 CM-treated C2C12 myotubes was decreased when STAT3 was knocked down by siRNA (Figure 6b). Next, we transfected a plasmid encoding constitutively activated STAT3 (STAT3-C) (Bromberg et al., 1999) into C2C12 myotubes. As expected, STAT3-C transfection significantly up-regulated MuRF-1, Atrogin-1, and myostatin, which was like that observed upon C26 CM treatment. Immunofluorescence staining confirmed that both STAT3-C, and C26 CM were able to increase the nuclear expression of FOXO1 (Figure 6c). We next sought to determine whether STAT3 could regulate FOXO1 transcription by direct binding of its promoter. According to the newest version of the JASPAR database (Fornes et al., 2020), two potential STAT3 DNA binding motifs in the promoter region of the FOXO1 gene were predicted (Figure 6d); and validated by chromatin immunoprecipitation assays. As shown in Figure 6e and f, semi-quantitative PCR and qPCR analysis of the DNA immunoprecipitated with the STAT antibody showed the enrichment of the FOXO1 promoter region sequence, indicating direct binding. In contrast, knocking down FOXO1 almost completely abolished STAT3 activation-induced myotube atrophy in both STAT3-C-transfected and C26 CM-treated C2C12 myotube cells (Figure 6g). Our data demonstrated for the first time that in cachectic skeletal muscle, consistent activation of STAT3 could up-regulate ubiquitin-ligases responsible for skeletal muscle protein degradation, MuRF1, and Atrogin1 in a FOXO1 pathway-dependent manner. Notably, the effect of 17DMAG on FOXO1 inhibition was abolished by the overexpression of STAT-C (Figure 6h), confirmed that FOXO1 was regulated by STAT3 at the transcription level.