Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

Abstract

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

Enhancement of learning and memory after activation of cerebral Rho GTPases

“…This consideration implies the potential pharmacological importance of the molecule. Because all experiments were performed between 10 and 28 days after a single injection, it seems conceivable a prolonged effect of the toxin on behavioral and electrophysiology parameters, which would be in accordance with the prolonged effect of CNF1 in peripheral tissues (51) and with its known mechanism of action. CNF1 can thus be regarded as endowed with long-lasting cognition enhancing properties.…”

“…This assay was conducted immediately after collection (4 h and 10 and 28 days after injection) of fresh brain left hemispheres (controlateral to the injection site). Homogenized samples were processed for pull-down assay as described (51). Details are provided in SI Methods.…”

Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of the Id3 Gene

“…Multiple Functions of RhoA/ ROCK Signaling in Myogenic Cells-The Rho family of small GTPases is involved in a variety of cellular events, including skeletal muscle differentiation (44). RhoA signaling is reported to be a positive regulator of myogenic differentiation, because the inactivation of RhoA decreases the expression of MyoD and inhibits myogenic differentiation (8).…”

Cytotoxic Necrotizing Factor 1 Prevents Apoptosis via the Akt/IκB Kinase Pathway: Role of Nuclear Factor-κB and Bcl-2

“…Pull-down assays were performed as described previously (Travaglione et al, 2005). Cells were lysed in 1) 50 mM HEPES, pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 0.5 M NaCl, and 10 mM MgCl 2 , plus protease inhibitors (to detect Rho-GTP) or 2) 50 mM HEPES, pH 7.4, 0.1 M NaCl, 10 mM MgCl 2 , 5% glycerol, 1% NP-40, and 10 mM NaF, plus protease inhibitors (to detect Rac/Cdc42-GTP).…”

“…Cells were lysed in 1) 50 mM HEPES, pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 0.5 M NaCl, and 10 mM MgCl 2 , plus protease inhibitors (to detect Rho-GTP) or 2) 50 mM HEPES, pH 7.4, 0.1 M NaCl, 10 mM MgCl 2 , 5% glycerol, 1% NP-40, and 10 mM NaF, plus protease inhibitors (to detect Rac/Cdc42-GTP). The cleared lysates were incubated with 80 g of glutathione S-transferase (GST)-Rhotekin (for Rho; Cytoskeleton, Denver, CO) and GST-PAK-CD (for Rac/Cdc42, prepared as described previously; Travaglione et al, 2005) fusion proteins, bound to glutathione-coupled Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) for 40 min at 4°C. Beads were washed three times in 1) 50 mM HEPES, pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 0.25 M NaCl, 5 mM MgCl 2 , plus protease inhibitors (for Rho) or 2) 50 mM HEPES, pH 7.4, 0.1 M NaCl, 10 mM MgCl 2 , 5% glycerol, 0.5% NP-40, and 10 mM NaF, plus protease inhibitors (for Rac/Cdc42).…”