RNAi screen reveals potentially novel roles of cytokines in myoblast differentiation.

Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases.

Flt3L is a novel regulator of skeletal myogenesis.

Various cues initiate multiple signaling pathways to regulate the highly coordinated process of skeletal myogenesis. Myoblast differentiation comprises a series of ordered events starting with cell cycle withdrawal and ending with myocyte fusion, with each step probably controlled by multiple extracellular signals and intracellular signaling pathways. Here we report the identification of Fms-like tyrokine kinase 3 ligand (Flt3L) signaling as a novel regulator of skeletal myogenesis. Flt3L is a multifunctional cytokine in immune cells, but its involvement in skeletal muscle formation has not been reported. We found that Flt3L is expressed in C2C12 myoblasts, with levels increasing throughout differentiation. Knockdown of Flt3L, or its receptor Flt3, suppresses myoblast differentiation, which is rescued by recombinant Flt3L or Flt3, respectively. Differentiation is not rescued, however, by recombinant ligand when the receptor is knocked down, or vice versa, suggesting that Flt3L and Flt3 function together. Flt3L knockdown also inhibits differentiation in mouse primary myoblasts. Both Flt3L and Flt3 are highly expressed in nascent myofibers during muscle regeneration in vivo, and Flt3L siRNA impairs muscle regeneration, validating the physiological significance of Flt3L function in myogenesis. We have identified a cellular mechanism for the myogenic function of Flt3L, as we show that Flt3L promotes cell cycle exit that is necessary for myogenic differentiation. Furthermore, we identify Erk as a relevant target of Flt3L signaling during myogenesis, and demonstrate that Flt3L suppresses Erk signaling through p120RasGAP. In summary, our work reveals an unexpected role for an immunoregulatory cytokine in skeletal myogenesis and a new myogenic pathway.

ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera.

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.