GPR97-Gα12-mTORC1 signaling drives myotube hypertrophy: identification of 5-hydroxy-7-methoxyflavone as a pathway activator.

Elucidating novel signaling pathways that drive myotube hypertrophy may provide new insights into the mechanisms regulating skeletal muscle growth. We previously reported that 5-hydroxy-7-methoxyflavone (HMF) induces hypertrophy of murine C2C12 myotubes; however, the underlying molecular mechanism remains unclear. Here, loss- and gain-of-function analyses revealed that GPR97 positively regulates myotube size. Furthermore, knockdown and rescue experiments demonstrated that GPR97 is required for HMF-induced myotube hypertrophy. HMF activated mTORC1 signaling in myotubes, and intramuscular administration of HMF also activated mTORC1 signaling in mouse skeletal muscle in vivo. Gpr97 knockdown abolished HMF-induced mTORC1 signaling and protein synthesis. Furthermore, depletion of Gna12 and expression of dominant-negative Gα12 abolished HMF-induced myotube hypertrophy. Gna12 depletion also suppressed HMF-induced mTORC1 signaling and protein synthesis. Beclomethasone dipropionate, a reported GPR97 ligand, suppressed HMF-induced hypertrophy and mTORC1 signaling, while further enhancing HMF-induced serum response factor (SRF)-dependent transcription. Although dominant-negative RhoA inhibited HMF-induced SRF-dependent transcriptional activity, it did not affect myotube hypertrophy. These findings indicate that GPR97-Gα12-mTORC1 signaling promotes hypertrophy independent of the Gα12-RhoA-SRF pathway. GPR97 was predominantly expressed on the myotube surface as a C-terminal fragment generated by N-terminal fragment (NTF) cleavage, and HMF reduced its surface expression. HMF induced hypertrophy in myotubes expressing an NTF cleavage-resistant GPR97 mutant, but not in those expressing an NTF-deleted mutant. These results indicate that NTF cleavage is dispensable and suggest that NTF contributes to GPR97-mediated hypertrophic signaling. These findings identify a novel GPR97-Gα12-mTORC1 signaling axis that mediates HMF-induced myotube hypertrophy.