Sophie Charrasse

N-cadherin–dependent cell–cell contact regulates Rho GTPases and β-catenin localization in mouse C2C12 myoblasts

N-cadherin, a member of the Ca2+-dependent cell–cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin–dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin–mediated signals involved in myogenesis, we investigated whether N-cadherin–dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin–dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin–mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for β-catenin accumulation at cell–cell contact sites. We propose that cell–cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.

A Cell Active Chemical GEF Inhibitor Selectively Targets the Trio/RhoG/Rac1 Signaling Pathway

RhoGEFs (guanine nucleotide exchange factors of the Rho GTPase family) are upstream regulators of cell adhesion and migration pathways, thus representing attractive yet relatively unexplored targets for the development of anti-invasive drugs. We screened for chemical inhibitors of TrioN, the N-terminal GEF domain of the multidomain Trio protein, and identified ITX3 as a nontoxic inhibitor. In transfected mammalian cells, ITX3 blocked TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on GEF337-, Tiam1-, or Vav2-mediated RhoA or Rac1 activation. ITX3 specifically inhibited endogenous TrioN activity, as evidenced by its ability to inhibit neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells or C2C12 differentiation into myotubes. This study introduces a selective cell active inhibitor of the Trio/RhoG/Rac1 pathway and validates RhoGEFs as druggable targets.

RhoE controls myoblast alignment prior fusion through RhoA and ROCK.

Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell–cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rho-associated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.

N-cadherin/P120 catenin association at cell-cell contacts occurs in cholesterol-rich membrane domains and is required for rhoa activation and myogenesis

p120 catenin is a major regulator of cadherin stability at cell-cell contacts and a modulator of Rho GTPase activities. In C2C12 myoblasts, N-cadherin is stabilized at cell contacts through its association with cholesterol-rich membrane domains or lipid rafts (LR) and acts as an adhesion-activated receptor that activates RhoA, an event required for myogenesis induction. Here, we report that association of p120 catenin with N-cadherin at cell contacts occurs specifically in LR. We demonstrate that interaction of p120 catenin with N-cadherin is required for N-cadherin association with LR and for its stabilization at cell contacts. LR disruption inhibits myogenesis induction and N-cadherin-dependent RhoA activation as does the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Finally, we observe an N-cadherin-dependent accumulation of RhoA at phosphatidylinositol 4,5-bisphosphate-enriched cell contacts which is lost after LR disruption. Thus, a functional N-cadherin-catenin complex occurs in cholesterol-rich membrane microdomains which allows the recruitment of RhoA and the regulation of its activity during myogenesis induction.

Variation in cadherins and catenins expression is linked to both proliferation and transformation of rhabdomyosarcoma

Cadherins are a family of transmembrane glycoproteins that mediate Ca2+-dependent homophilic cell–cell adhesion and play a crucial role in cell differentiation. E-cadherin-mediated cell–cell adhesion is lost during the development of most epithelial cancers. This study examines cadherin-dependent adhesion in cell lines derived from rhabdomyosarcoma (RMS), a highly malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We analysed the expression of cadherins and associated catenins at the mRNA and protein levels as well as their localization in nine RMS-derived cell lines relative to normal myoblasts. We show a decrease in the expression of cadherins and catenins in all RMS-derived cell lines compared to control cells. This decrease in the expression of N- and M-cadherin was confirmed in RMS biopsies. In contrast, R-cadherin is found expressed in RMS, whereas it is normally absent in normal myoblasts. We show that a decrease of R-cadherin expression using RNA interference inhibits cell proliferation of the RD cell line. In addition to their diminished expression, cadherins and catenins do not localize to intercellular contacts in embryonal RMS (ERMS), whereas specific persistent localization is seen in alveolar RMS (ARMS)-derived cell lines. Thus, RMS exhibit defects in the expression of molecules of the cadherin family. Defects in the localization of these adhesion molecules at the sites of cell–cell contact are specifically observed in the ERMS subtype. In addition, our data suggest that R-cadherin is a specific diagnostic marker for RMS and is also an important factor of RMS cell proliferation.

ADP-ribosylation factor 6 regulates mammalian myoblast fusion through phospholipase D1 and phosphatidylinositol 4,5-bisphosphate signaling pathways.

Here we show that ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through Phospholipase D activation and PI(4,5)P2 production.

Rho GTPases and cadherin-based cell adhesion in skeletal muscle development

The small GTPases of the Rho subfamily (RhoA, Rac1 and Cdc42) are signaling molecules involved in cytoskeleton remodeling and gene transcription. Their activities are important for many cellular processes, including myogenesis. Classical cadherin adhesion molecules are key determinants of cell recognition and tissus morphogenesis and act as adhesion-activated signaling receptors. Rho GTPases have emerged as key mediators of their activity. Not only signal transduction pathways link cadherins to Rho GTPases but also Rho GTPases to cadherins. We focus in this review on the role of cadherins and Rho GTPases in normal myogenesis as well as in pathological development of rhabdomyosarcoma.

Transforming growth factor β activates Rac1 and Cdc42Hs GTPases and the JNK pathway in skeletal muscle cells

Summry— The transforming growth factor β (TGFβ) plays an important role in cell growth and differentiation. However, the intracellular signaling pathways through which TGFβ inhibits skeletal myogenesis remain largely undefined. By measuring GTP‐loading of Rho GTPases and the organization of the F‐actin cytoskeleton and the plasma membrane, we analyzed the effect of TGFβ addition on the activity of three GTPases, Rac1, Cdc42Hs and RhoA. We report that TGFβ activates Rac1 and Cdc42Hs in skeletal muscle cells, two GTPases previously described to inhibit skeletal muscle cell differentiation whereas it inactivates RhoA, a positive regulator of myogenesis. We further show that TGFβ activates the C‐jun N‐terminal kinases (JNK) pathway in myoblastic cells through Rac1 and Cdc42Hs GTPases. We propose that the activation of Rho family proteins Rac1 and Cdc42Hs which subsequently regulate JNK activity participates in the inhibition of myogenesis by TGFβ.

Thyroid Hormone Promotes Transient Nerve Growth Factor Synthesis in Rat Cerebellar Neuroblasts

Primary cultures of cerebellum from 5-day-old rats indicated that proliferating neuroblasts synthesize and release nerve growth factor (NGF). Since NGF promotes DNA synthesis in these cells, our findings demonstrate that the early developing cerebellum is a suitable physiological model for studying the autocrine mitogenic action of NGF. Thyroid deficiency led to a greater reduction in the NGF content of the cerebellum than of the olfactory bulbs or hippocampus. Cerebellar NGF mRNA was also very sensitive to hormone deprivation. Physiological amounts of thyroid hormone stimulated both the mitotic activity and NGF production of cultured cerebellar neuroblasts. A lack of thyroid hormone is known to markedly alter cell formation in the cerebellum where postnatal neurogenesis is highly significant, in contrast to the olfactory bulbs and hippocampus. Taken together, these results suggest that the hormonal control of cell formation in the cerebellum is, at least partly, mediated by the autocrine mitogenic action of NGF. The thyroid hormone could temporally regulate the transient NGF synthesis by cerebellar neuroblasts directly and/or through its ontogenetic action, and hence all the NGF-dependent trophic effects.

Participation of small GTPases Rac1 and Cdc42Hs in myoblast transformation.

We have previously shown that expression of active Rac1 and Cdc4Hs inhibits skeletal muscle cell differentiation. We show here, by bromodeoxyuridine incorporation and cyclin D1 expression, that the expression of active Rac1 and Cdc42Hs but not RhoA impairs cell cycle exit of L6 myoblasts cultured in differentiation medium. Furthermore, expression of activated forms of Rac1 and Cdc42Hs elicits the loss of cell contact inhibition and anchorage-dependent growth as measured by focus forming activity and growth in soft agar. RhoA was once again not found to have this effect. We found a constitutive Rac1 and Cdc42Hs activation in three human rhabdomyosarcoma-derived cell lines, one of the most common causes of solid tumours arising from muscle precursors during childhood. Finally, dominant negative forms of Rac1 and Cdc42Hs inhibit cell proliferation of the RD rhabdomyosarcoma cell line. These data suggest an important role for the small GTPases Rac1 and Cdc42Hs in the generation of skeletal muscle tumours.