TRIM72, a novel negative feedback regulator of myogenesis, is transcriptionally activated by the synergism of MyoD (or myogenin) and MEF2
Introduction
Skeletal muscle fibers are classified as slow oxidative (type I), fast oxidative (type IIa and type IIx), and fast glycolytic fibers (type IIb) on the basis of their contraction speed, fatigue resistance, and myosin heavy chain (MHC) isoform expression. Gastrocnemius muscle, which is largely composed of type IIb fibers, is used during intense and rapid activities and has a high speed of contraction. Soleus muscle with a high proportion of types I and IIa fibers is used for postural tension and has a high fatigue resistance [1]. Exercise training or motor neuron activity remodels muscle fiber types in adult skeletal muscle [1], [2], [3], [4], [5].
Myocyte enhancer factor 2 (MEF2) transcriptionally activates muscle-specific genes with an A/T-rich consensus sequence (C/TTA(A/T)4TAG/A) called a MEF2 site [6]. MEF2 responds to calcium signaling pathways involved in muscle type remodeling. The number of oxidative fibers is reduced in the soleus of MEF2C- or MEF2D-disrupted mice [7], and exercise training increases β-galactosidase activity in the oxidative fibers of transgenic mice carrying the lacZ gene with MEF2 sites [8], [9], indicating that MEF2 regulates muscle type remodeling. The transcriptional activity of MEF2 is enhanced by Ca2+-induced calcineurin activation but inhibited by class II histone deacetylases (HDACs) [8], [10]. Calcium/calmodulin-dependent kinase (CaMK) and protein kinase D1 induce the phosphorylation and cytoplasmic translocation of HDACs, dissociating MEF2 from HDACs in the nucleus [11], [12], [13], [14].
TRIM72, with a RING finger domain, a B-box, two coiled-coil domains, and a SPRY domain is specifically expressed in the plasma membrane of skeletal and cardiac muscle cells [15], [16]. In addition, the expression level of TRIM72 gradually increases during C2C12 myogenesis. TRIM72 has been known to regulate skeletal muscle differentiation since its adenoviral overexpression prevents, but its knock-down enhances the myogenesis of C2C12 cells and human primary skeletal muscle cells [16]. Since MyoD-dependent TRIM72 promoter activity is regulated by the PI(3)K-Akt pathway, TRIM72 is highly expressed in the plasma membrane of C2C12 myotubes during IGF-mediated myogenesis. The accumulated TRIM72 in the plasma membrane interacts with insulin receptor substrate-1 (IRS-1) and inhibits the IGF-induced activation of IRS-1, blocking excess myogenesis [16]. Thus, TRIM72 might be a negative feedback regulator of myogenesis for regulating muscle fiber size.
In order to understand molecular mechanism of TRIM72 expression during skeletal muscle differentiation, we analyzed the transcriptional regulation of TRIM72, of which promoter contains a MEF site as well as five E-boxes. Here, we demonstrated that TRIM72 transcription was synergistically activated by MyoD (or myogenin), and MEF2 during C2C12 skeletal muscle differentiation, thereby explaining why soleus oxidative muscle with higher activity of MEF2 has a higher expression level of TRIM72 than gastrocnemius glycolytic muscle does.
Section snippets
Antibodies and siRNAs
Rabbit anti-TRIM72 polyclonal antibody was generated from AbFrontier. Anti-MyoD (sc-760X), myogenin (sc-576X), and MEF2 (sc-313X) antibodies used for ChIP were purchased from Santa Cruz biotechnology, and anti-MyoD (554130) and myogenin (556358) antibodies used for EMSA from BD Transduction laboratories. Small interference RNA oligonucleotides for control (si-Con) and myogenin (si-Mgn) were purchased from Santa Cruz Biotechnology.
Animals and Western blotting
C57BL/6 mice were housed in plastic cages on a 12:12 h light–dark
TRIM72 promoter contains an evolutionarily conserved MEF2 site
Oxidative fibers have smaller diameters than glycolytic fibers do and therefore the diffusion distance for oxygen to travel to the mitochondria is reduced. Since TRIM72 is proposed to be a negative size regulator in skeletal muscle [16], it is tempting to speculate that TRIM72 might be highly expressed in oxidative fibers. In order to address the issue, TRIM72 expression levels were determined in mouse soleus, tibialis anterior (TA), and gastrocnemius muscles by Western blotting. Among the
Acknowledgments
This work was supported by grants to Y.-G. Ko from Korea Research Foundation (KRF-2007-313-C00582).
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