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.