Characterization of a dominant inhibitory E47 protein that suppresses C2C12 myogenesis.

Skeletal muscle formation is controlled through the coordinated actions of the muscle regulatory factors (MRFs). The activities of these basic helix-loop-helix proteins is mediated in part through heterodimer formation with a family of ubiquitous bHLH proteins, referred to as E-proteins. The primary E-protein in skeletal muscle is the E2A splice variant, E47. To further address the role of E47 during skeletal myogenesis, we created a chimeric E47 repressor protein by replacing the transcriptional activation domain with the Drosophila Engrailed transcriptional repressor domain. The dominant inhibitory E-protein (EnDeltaE47) formed homodimers capable of binding DNA and abolished E47-directed gene transcription. Stable expression of EnDeltaE47 in mouse C2C12 myoblasts effectively blocked the cells' ability to differentiate into mature myofibers. Closer examination of the molecular basis for the inhibition of myogenesis revealed that EnDeltaE47 preferentially forms heterodimers with myogenin. Interestingly, the chimeric repressor did not form DNA-binding heterodimers with MyoD in C2C12 myocytes. The failure to detect MyoD:EnDeltaE47 heterodimers in myoblasts was not due to protein conformational defects as both wild-type E47 and EnDeltaE47 readily formed DNA binding complexes with MyoD in vitro. These results indicate that E47 plays a crucial role in C2C12 myogenesis by serving as the preferred heterodimer partner of the myogenin protein.

FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb.

FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS-FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin-expressing, connective-tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked-cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle.

Activated Raf inhibits myogenesis through a mechanism independent of activator protein 1-mediated myoblast transformation.

Skeletal myogenesis is acutely affected by growth factors and subsequent activation of their respective intracellular signaling cascades. Components of the mitogenic Ras/Raf/mitogen-activated protein kinase (MAPK) signaling module are potent inhibitors of myoblast differentiation. However, the means by which these kinases prevent myocyte formation and activation of the muscle gene program is unknown. Activator protein 1 (AP-1) is a transcriptional regulator the actions of which are up-regulated by signaling events, including elevated MAPK. Because activated Raf inhibits avian myogenesis in a MAPK-dependent fashion, we investigated the role of AP-1 as a mediator of the Raf-imposed block to myogenesis. Avian myoblasts overexpressing activated Raf contain elevated levels of AP-1 DNA binding and transcriptional activity. Introduction of an AP-1 dominant inhibitory protein (AFOS) into Raf-expressing myoblasts prevented acquisition of a transformed morphology. Interestingly, these cells remained differentiation-defective. Myogenic cells cotransduced with RCAS(A)-Raf BXB and RCAS(B)-AFOS remained mononuclear and myosin-negative and did not activate significantly muscle-specific reporter genes. These results argue that Raf inhibits muscle differentiation independent of AP-1-mediated cell transformation. Our results provide evidence for AP-1 as a critical component of the transforming capacity of activated Raf and evidence that AP-1 is not involved in the myogenic inhibitory effects of the kinase.

Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism.

Chronic overexpression of the oncogenic form of Ras is a potent inhibitor of skeletal myogenesis. However, the intracellular signaling pathways that mediate the repressive actions of Ras on myogenic differentiation have yet to be identified. We examined the role of Raf-mediated signaling as a modulator of avian myogenesis. Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes. Most notably, the embryonic chick myoblasts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or RCAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures. While residual myofibers were apparent in the RCAS-Raf BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic phenotype. The altered morphology is not a result of reinitiation of the myonuclei cell cycle nor is it due to apoptosis. Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity. Supplementation of the culture media with the MAPK kinase (MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls. Our results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to myogenesis.

High throughput assays of cloned adrenergic, muscarinic, neurokinin, and neurotrophin receptors in living mammalian cells.

Many receptors stimulate proliferation of NIH 3T3 cells in a ligand dependent fashion. Based on this observation, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme beta-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. In the present study, we used this assay to evaluate the ability of agonist ligands to stimulate four cloned receptors. The agonists phenylephrine, carbachol, substance P and nerve growth factor selectively stimulated cells transfected with the alpha-1b adrenergic, m4 muscarinic, NK1 neurokinin and trkA neurotrophin receptors, respectively. These data demonstrate that a high throughput colorimetric assay performed in 96 well plates can be used to evaluate the pharmacology of ligands for cloned receptors belonging to a wide range of functional and pharmacological classes.