Abstract
Most recent studies reported that FoxO1 transcription factor was a negative regulator of myogenesis under serum withdrawal condition, a situation not actually found in vivo. Therefore, the role of FoxO1 in myogenesis should be re-examined under more physiologically relevant conditions. Here we found that FoxO1 was preferentially localized to nucleus in proliferating (PMB) and confluent myoblasts (CMB) and its nuclear exclusion was a prerequisite for formation of multinucleated myotubes (MT). The nuclear shuttling of FoxO1 in PMB could be prevented by leptomycin B and we further found that cytoplasmic accumulation of FoxO1 in myotubes was caused by the blockade of its nuclear import. Although over-expression of wildtype FoxO1 in C2C12 myoblasts significantly blocked their myogenic differentiation under serum withdrawal condition, application of insulin and LiCl, an activator of Wnt signaling pathway, to these cells successfully rescued their myogenic differentiation and generated myotubes with larger diameters. Interestingly, insulin treatment significantly reduced FoxO1 level and also delayed nuclear re-accumulation of FoxO1 triggered by mitogen deprivation. We further found that FoxO1 directly repressed the promoter activity of myogenic genes and this repression can be relieved by insulin and LiCl treatment. These results suggest that FoxO1 inhibits myogenesis in serum withdrawal condition but turns into a hypertrophy potentiator when other myogenic signals, such as Wnt and insulin, are available.
Highlights
Transcription factors of the FoxO family, including FoxO1 (FKHR), FoxO3 (FKHRL1), FoxO4 (AFX), and FoxO6, have been discovered to play important roles in a diverse sets of cellular physiological functions [1,2,3]
To examine the function of FoxO1 in myogenic differentiation, we started with the over-expression of FoxO1 in C2C12 myoblasts by infecting them with retrovirus carrying wildtype or constitutively active (AAA, in which the 3 Akt sites were mutated to alanine) form of FoxO1 coding sequence and selected with antibiotics (G418) for 2–3 weeks to generate stable clones (C2C12FoxO1) expressing FoxO1
The effect of FoxO1 over-expression on C2C12 moygenic differentiation was tested by serum withdrawal induced myogenic differentiation, in which both control and C2C12-FoxO1 cells grown to confluent were induced to differentiate by changing to differentiation medium (DM) containing 2% horse serum
Summary
Transcription factors of the FoxO family, including FoxO1 (FKHR), FoxO3 (FKHRL1), FoxO4 (AFX), and FoxO6, have been discovered to play important roles in a diverse sets of cellular physiological functions [1,2,3] They can function by direct binding to DNA or by tethered to the target site through protein-protein interaction with other transcription factors, such as nuclear receptors and HNF4, [4]. Phosphorylated FoxO1 tends to be shuttled out of nucleus and lost their binding to target regulatory elements [6,7] These 3 Akt targeted sites, named as T1, S1, and S2, are conserved from Daf in C. elegans to its orthologs in mammals [8]. In addition to these Akt-targeted sites, multiple residues in FoxO1 are phosphoryalted by other kinases, including CDK2, DYRK1 and CK1 [9,10,11]
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