MEHP interferes with mitochondrial functions and homeostasis in skeletal muscle cells.

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.

Phosphorylation of adducin-1 by cyclin-dependent kinase 5 is important for epidermal growth factor-induced cell migration.

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neuron and plays an important role in neuronal physiology. Increasing evidence also indicates that Cdk5 may contribute to malignant progression of some types of cancers; however, the underlying mechanism remains elusive. In this study, we found that Cdk5 directly phosphorylated the actin-binding protein adducin-1 (ADD1) at T724 in vitro and in intact cells. The capability of the phosphomimetic T724D mutant to bind to actin filaments was lower than that of wild type ADD1 and the T724A mutant. Cdk5 co-localized with ADD1 at the lamellipodia upon epidermal growth factor (EGF) stimulation. The increased lamellipodia formation and cell migration of human breast cancer cells MDA-MB-231 by EGF were accompanied by Cdk5 activation and increased phosphorylation of ADD1 at T724. Depletion of Cdk5 in MDA-MB-231 cells abrogated the effects of EGF on ADD1 T724 phosphorylation, lamellipodia formation, and cell migration. Likewise, depletion of ADD1 suppressed the effects of EGF on lamellipodia formation, cell migration, and invasion, all of which were restored by FLAG-ADD1 WT and the T724D mutant, but not the T724A mutant. Together, our results suggest that phosphorylation of ADD1 at T724 by Cdk5 is important for EGF-induced cell migration and invasion.

Correction: Src and SHP2 coordinately regulate the dynamics and organization of vimentin filaments during cell migration.

The original version of this article contained error in Fig. 6b, where several panels were missing from the published version. The corrected version of this Figure now appears in the article.

Src and SHP2 coordinately regulate the dynamics and organization of vimentin filaments during cell migration.

Vimentin intermediate filaments (VIFs), expressed in most mesenchymal and cancer cells, undergo dramatic reorganization during cell migration; however, the mechanism remains obscure. This study demonstrates that upon growth-factor stimulation, Src directly phosphorylates vimentin at Tyr117, leading to VIF disassembly into squiggles and particles at the cell edge during lamellipodia formation. The protein tyrosine phosphatase SHP2 counteracted the Src effects on VIF tyrosine phosphorylation and organization. VIFs formed by vimentin Y117D mutant were more soluble and dynamic than those formed by the wild-type and Y117F mutant. Increased expression of vimentin promoted growth-factor induced lamellipodia formation and cell migration, whereas the mutants suppressed both. The vimentin-induced increase in lamellipodia formation correlated with the activation of Rac and Vav2, with the latter associated with VIFs and recruited to the plasma membrane upon growth-factor stimulation. These results reveal a novel mechanism for regulating VIF dynamics through Src and SHP2 and demonstrate that proper VIF dynamics are important for Rac activation and cell migration.

Bhlhe40 differentially regulates the function and number of peroxisomes and mitochondria in myogenic cells.

PGC-1α is a key regulator of oxidative metabolism facilitating the expression of genes critical for the function and biogenesis of the two key oxidative organelles, mitochondria and peroxisomes, in skeletal muscle (SKM) and other organs. Our recent studies have found that the transcription factor Bhlhe40 negatively regulates PGC-1α gene expression and its coactivational activity, therefore, this factor should have profound influence on the biogenesis and metabolic activity of mitochondria and peroxisomes. Here we found that both the number and activity of peroxisomes were increased upon knockdown of Bhlhe40 expression but were repressed by its over-expression. Mitochondrial efficiency was significantly reduced by Bhlhe40 knockdown, resulting in the burst of ROS. Over-expression of a constitutively active PGC-1α-interactive domain (named as VBH135) of Bhlhe40 mimicked the effects of its knockdown on peroxisomes but simultaneously reduced ROS level. Furthermore, the efficiency, but not the number, of mitochondria was also increased by VBH135, suggesting differential regulation of peroxisomes and mitochondria by Bhlhe40. Unsaturated fatty acid oxidation, insulin response, and oxidative respiration were highly enhanced in Bhlhe40 knockdown or VBH135 over-expressed cells, suggesting the importance of Bhlhe40 in the regulation of unsaturated fatty acid and glucose oxidative metabolism. Expression profiling of genes important for either organelle also supports differential regulation of peroxisomes and mitochondria by Bhlhe40. These observations have established the important role of Bhlhe40 in SKM oxidative metabolism as the critical regulator of peroxisome and mitochondrion biogenesis and functions, and thus should provide a novel route for developing drugs targeting SKM metabolic diseases.

Bhlhe40 Represses PGC-1α Activity on Metabolic Gene Promoters in Myogenic Cells.

PGC-1α is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1α might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1α directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1α-targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity. Bhlhe40 repressed PGC-1α activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1α intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1α target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1α-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1α-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1α activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM.

Wnt3a signal pathways activate MyoD expression by targeting cis-elements inside and outside its distal enhancer.

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 ß-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.