Insulin-like growth factor-I downregulates embryonic myosin heavy chain (eMyHC) in myoblast nuclei

Abstract

The obscure ability of the insulin-like growth factors (IGF-I & -II) to stimulate both myoblast proliferation and differentiation suggests that the latter effect may be mediated locally, possibly by IGF binding proteins (IGFBPs). In some cells, the growth inhibitory actions of IGFBP-5 require plasma membrane translocation and nuclear localization. Immunoreactivity of presumably endogenous IGFBP-5 was identified within proliferating rat L6 myoblast nuclei using fluorescent and confocal microscopy in separate experiments and was reduced by 100 ng/ml IGF-I in a time-dependent manner. Western blotting of nuclear and cytosolic protein identified a single anti-IGFBP-5 immunoreactive protein of approximately 200 kDa, primarily in nuclear fractions, that was downregulated in cells treated with IGF-I for 12 h. The unknown protein was immunopurified from nuclear fractions and identified as the rat homologue for embryonic myosin heavy chain (eMyHC) using matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy. Cross-reactivity of the IGFBP-5 antiserum with eMyHC was confirmed by blotting anti-IGFBP-5 nuclear immunoprecipitates with eMyHC monoclonal antibodies (F1.652). These data indicate that eMyHC is located predominantly within the nuclei of proliferating L6 myoblasts and suggest that IGF-stimulated differentiation is associated with the rapid downregulation of nuclear eMyHC as these cells stop expressing this myosin II isoform as they differentiate. Myosin Iβ has been identified within the nuclei of non-muscle cells where it helps to regulate gene transcription. Thus, eMyHC may serve a similar role in myoblasts that is specific only to the undifferentiated state.