ABSTRACT
INTRODUCTION: Skeletal muscle tissue development and regeneration relies on the proliferation, maturation and fusion of muscle progenitor cells (myoblasts), which arise transiently from muscle stem cells (satellite cells). Following muscle damage, myoblasts proliferate and differentiate in response to temporally-varying inflammatory cytokines, growth factors, and extracellular matrix cues, which stimulate a shared network of intracellular signaling pathways. Here we present an integrated data-modeling approach to elucidate synergies and antagonisms among proliferation and differentiation signaling axes in myoblasts stimulated by regeneration-associated ligands. METHODS: We treated mouse primary myoblasts in culture with combinations of eight regeneration-associated growth factors and cytokines in mixtures that induced additive, synergistic, and antagonistic effects on myoblast proliferation and differentiation responses. For these combinatorial stimuli, we measured the activation dynamics of seven signal transduction pathways using multiplexed phosphoprotein assays and scored proliferation and differentiation responses based on expression of myogenic commitment factors to assemble a cue-signaling-response data compendium. We interrogated the relationship between these signals and responses by partial least-squares (PLS) regression modeling. RESULTS: Partial least-squares data-modeling accurately predicted response outcomes in cross-validation on the training compendium (cumulative R 2 = 0.96). The PLS model highlighted signaling axes that distinctly govern myoblast proliferation (MEK-ERK, Stat3) and differentiation (JNK) in response to these combinatorial cues, and we confirmed these signal-response associations with small molecule perturbations. Unexpectedly, we observed that a negative feedback circuit involving the phosphatase DUSP6/MKP-3 auto-regulates MEK-ERK signaling in myoblasts. CONCLUSION: This data-modeling approach identified conflicting signaling axes that underlie muscle progenitor cell proliferation and differentiation.
