Wnt proteins are secreted cytokines and several Wnts are expressed in the developing somites and surrounding tissues. Without proper Wnt stimulation, the organization of the dermomyotome and myotome can become defective. These Wnt signals received by somitic cells can lead to activation of Pax3/Pax7 and myogenic regulatory factors (MRFs), especially Myf5 and MyoD. However, it is currently unknown whether Wnts activate Myf5 and MyoD through direct targeting of their cis-regulatory elements or via indirect pathways. To clarify this issue, in the present study, we tested the regulation of MyoD cis-regulatory elements by Wnt3a secreted from human embryonic kidney (HEK)-293T cells. We found that Wnt3a activated the MyoD proximal 6.0k promoter (P6P) only marginally, but highly enhanced the activity of the composite P6P plus distal enhancer (DE) reporter through canonical and non-canonical pathways. Further screening of the intervening fragments between the DE and the P6P identified a strong Wnt-response element (WRE) in the upstream −8 to −9k region (L fragment) that acted independently of the DE, but was dependent on the P6P. Deletion of a Pax3/Pax7-targeted site in the L fragment significantly reduced its response to Wnt3a, implying that Wnt3a activates the L fragment partially through Pax3/Pax7 action. Binding of β-catenin and Pax7 to their target sites in the DE and the L fragment respectively was also demonstrated by ChIP. These observations demonstrated the first time that Wnt3a can directly activate MyoD expression through targeting cis-elements in the DE and the L fragment.

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.

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer frequently leached out from polyvinyl chloride (PVC) products and is quickly metabolized to its monoester equivalent mono(2-ethylhexyl) phthalate (MEHP) once enters organisms. Exposure to DEHP/MEHP through food chain intake has been shown to modified metabolism but its effect on the development of metabolic myopathy of skeletal muscle (SKM) has not been revealed so far. Here, we found that MEHP repressed myogenic terminal differentiation of proliferating myoblasts (PMB) and confluent myoblasts (CMB) but had weak effect on this process once it had been initiated. The transition of mitochondria (MITO) morphology from high efficient filamentary network to low efficient vesicles was triggered by MEHP, implying its negative effects on MITO functions. The impaired MITO functions was further demonstrated by reduced MITO DNA (mtDNA) level and SDH enzyme activity as well as highly increased reactive oxygen species (ROS) in cells after MEHP treatment. The expression of metabolic genes, including PDK4, CPT1b, UCP2, and HO1, was highly increased by MEHP and the promoters of PDK4 and CPT1b were also activated by MEHP. Additionally, the stability of some subunits in the oxidative phosphorylation system (OXPHOS) complexes was found to be reduced by MEHP, implying defective oxidative metabolism in MITO and which was confirmed by repressed palmitic acid oxidation in MEHP-treated cells. Besides, MEHP also blocked insulin-induced glucose uptake. Taken together, our results suggest that MEHP is inhibitory to myogenesis and is harmful to MITO functions in SKM, so its exposure should be avoided or limited.