Abstract
To investigate the contribution of individual arginines, we employed site-directed mutagenesis to generate arginine-to-alanine (R→A) substitution mutations in the N-terminal domain of Forkhead box M1 (FoxM1). The R15A mutation impaired FoxM1 transcriptional activity, hindered FoxM1 nuclear translocation and failed to promote the migratory and invasive behavior of glioma cells than other single arginine mutations. Furthermore, we demonstrated that FoxM1 expression was associated with Mitochondrial transcription factor A (TFAM) expression. Overexpressing FoxM1 increased TFAM protein levels, which was reversed by FoxM1 knockdown in glioblastoma multiforme (GBM) cells. The siRNA-mediated reduction of TFAM expression was rescued by FoxM1 overexpression. Also, FoxM1 overexpression promoted TFAM promoter luciferase activity. Importantly, the R15A mutation failed to promote TFAM expression. Additionally, FoxM1 increased the expression of mitochondrial fusion markers, Optic atrophy protein 1 (OPA1) and Mitofusin 1 (MFN1) and led to interconnected mitochondria, while FoxM1 knockdown reversed this effect. Moreover, FoxM1 promoted mitochondrial fission markers, Dynamin-related protein 1 (DRP1), Mitochondrial fission factor (MFF) and Mitochondrial fission protein 1 (FIS1). Notably, the R15A mutation resulted in loss of FoxM1 regulation of fusion and fission-related protein expression. Taken together, our findings reveal that that the N-terminal arginine 15 is a key site for the transcriptional activation and function of FoxM1 in GBM cells, suggesting its potential as a therapeutic target in GBM.
Keywords:
FoxM1; N-terminal arginine residues, GBM; TFAM; mitochondrial fusion/fission.