Transforming Growth Factor β (TGF-β) Signaling Is Regulated by Electrical Activity in Skeletal Muscle Cells

Abstract

Transforming growth factor (TGF-␤) is involved in several cellular processes such as cell proliferation, differentiation, and apoptosis. At the cell surface, TGF-␤ binds to serine-threonine kinase transmembrane receptors (type II and type I) to initiate Smad-dependent intracellular signaling cascades. During the early stages of skeletal muscle differentiation, myotubes start to evoke spontaneous electrical activity in association with contractions that arise following the maturation of the excitation-contraction apparatus. In this work, we report that TGF-␤-dependent signaling is regulated by electrical activity in developing rat primary myotubes, as determined by Smad2 phosphorylation, Smad4 nuclear translocation, and p3TPLux reporter activity. This electrical activity-dependent regulation is associated with changes in TGF-␤ type I receptor (T␤RI) levels, correlated with changes in transducing receptors at the cell membrane (measured through radiolabeling binding assays). The inhibition of electrical activity with tetrodotoxin, a voltage-dependent sodium channel blocker, increases T␤RI levels via a transcription-dependent mechanism. In contrast, the promotion of electrical activity in myotube cultures, induced by the upregulation of voltage-dependent sodium channels or by direct stimulation with extracellular electrodes, causes T␤RI levels to decrease. Similar results were obtained in denervated adult muscles, suggesting that electrical activity-dependent regulation of T␤RI also occurs in vivo. Additional results suggest that this activitydependent regulation is mediated by myogenin. Altogether, these findings support the possibility for a novel regulatory mechanism acting on TGF-␤ signaling cascade in skeletal muscle cells.

Syndecan-4 and β1 integrin are regulated by electrical activity in skeletal muscle: Implications for cell adhesion

“…By contrast, in the denervated muscle TβRI is up-regulated as previously reported (Fig. 4B) (Ugarte and Brandan, 2006). This result indicates that the inhibition of muscle activity can also affect syndecan-4 levels in vivo.…”

“…Fig. 5B shows that protein levels of β1 integrin after the inhibition of electrical activity for 24 h increase progressively from 6 h after restoring the spontaneous activity and almost reaches control levels after 24 h. The lower panel shows that the recovery of β1 integrin protein levels is accompanied of the down-regulation of TβRI, which is negatively regulated by spontaneous electrical activity (Ugarte and Brandan, 2006).…”

“…Under culture conditions, rat primary myotubes start to develop spontaneous electrical activity associated to visible contractile activity at day 2 of differentiation (Dutton et al, 1993;Ugarte and Brandan, 2006). To determine whether the amount of syndecan-4 is regulated by electrical activity in primary cultures, this activity was inhibited by the addition of TTX to culture media.…”

“…Protein concentrations were determined using the Micro BCA™ protein assay kit (Pierce, Rockford, IL, USA). Samples were electrophoresed in 10% polyacrylamide gels (or in 4-12% gradient gels for separate HSPGs), transferred onto nitrocellulose membranes and probed with one of the following antibodies: mouse anti-Δ-heparan sulfate 3G10 (1:2500) (USBiological, Swampscott, MA, USA); rabbit anti-TGF-β type I receptor (TβRI) (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-β1 integrin (1:1000) (Santa Cruz Biotechnology); rabbit antiphospho-Akt (Ser 473) (1:1000) (Cell Signaling Technology, Beverly, MA, USA); rabbit anti-Akt (1:1000) (Cell Signaling Technology); rabbit anti-alpha 5 integrin (1:1000) (Santa Cruz Biotechnology); rabbit anti-phosphorylated Smad-2 (1:500) (Calbiochem, San Diego, CA, USA); or mouse anti-GADPH (1:5000) (Chemicon, Temecula, CA, USA) (Fuentealba et al, 1999;Ugarte and Brandan, 2006;Cabello-Verrugio and Brandan, 2007;Vial et al, 2008). After incubation with HRPconjugated secondary antibodies (1:10,000) (Pierce, Rockford, IL, USA), reactive proteins were visualized using chemiluminescent substrates (Pierce, Rockford, IL, USA).…”

Activity-dependent gene regulation in skeletal muscle is mediated by a histone deacetylase (HDAC)-Dach2-myogenin signal transduction cascade

“…We used pharmacological inhibition of HDAC's in C2C12 myotubes and adult muscle fibers to show HDAC activity is necessary for Mgn, nAChR, and MuSK gene induction in inactive muscle. Although HDAC inhibitors have previously been used to study myogenesis (26) and have been shown to inhibit Mgn expression in differentiated myotubes (27,28), their effect on adult skeletal muscle has not been reported. Our finding that both mature myotubes and adult denervated muscle require HDAC activity and Dach2 repression for Mgn and Mgn-dependent gene induction suggests a common underlying mechanism.…”