ABSTRACT
DNA topoisomerase I (Topo1) contributes to vital biological functions, but its regulation is not clearly understood. The BTBD1 protein was recently cloned on the basis of its interaction with the core domain of Topo1 and is expressed particularly in skeletal muscle. To determine BTBD1 functions in this tissue, the in vitro model used was the C2C12 mouse muscle cell line, which expresses BTBD1 mainly after myotube differentiation. We studied the effects of a stably overexpressed BTBD1 protein truncated of the 108 N-terminal amino-acid residues and harbouring a C-terminal FLAG tag (Delta-BTBD1). The proliferation speed of Delta-BTBD1 C2C12 cells was significantly decreased and no myogenic differentiation was observed, although these cells maintained their capacity to enter adipocyte differentiation. These alterations could be related to Topo1 deregulation. This hypothesis is further supported by the decrease in nuclear Topo1 content in Delta-BTBTD1 proliferative C2C12 cells and the switch from the main peripheral nuclear localization of Topo1 to a mainly nuclear diffuse localization in Delta-BTBTD1 C2C12 cells. Finally, this study demonstrated that BTBD1 is essential for myogenic differentiation.
Subject(s)
DNA-Binding Proteins/physiology , Muscles/cytology , Transcription Factors/physiology , Adipocytes/cytology , Adipocytes/metabolism , Animals , Azo Compounds/pharmacology , Blotting, Northern , Blotting, Western , Cell Differentiation , Cell Line , Cell Proliferation , Coloring Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , DNA, Complementary/metabolism , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Muscle, Skeletal/cytology , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA/metabolism , Time Factors , TransfectionABSTRACT
C2C12 cells are a well-established model system for studying myogenesis. We examined whether inhibiting the process of myogenesis via expression of dominant negative (DN) mitogen-activated protein kinase kinase-3 (MKK3) facilitated the trans-differentiation of these cells into adipocytes. Cells expressing DN MKK3 respond to rosiglitazone, resulting in adipocyte formation. The effects of rosiglitazone appear to be potentiated through peroxisome proliferator activating receptor-gamma. This trans-differentiation is inhibited by the use of the phosphoinositide-3 (PI3) kinase inhibitor, LY294002. These results indicate that preventing myogenesis through expression of DN MKK3 facilitates adipocytic trans-differentiation, and involves PI3 kinase signalling.
Subject(s)
Adipocytes/physiology , Cell Differentiation/physiology , Muscle Development , Thiazolidinediones , Adipocytes/cytology , Biomarkers , Cell Line , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , MAP Kinase Kinase 3 , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase Kinases/metabolism , Morpholines/pharmacology , Muscles/cytology , Muscles/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein-Tyrosine Kinases/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Rosiglitazone , Thiazoles/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Vasodilator Agents/pharmacologyABSTRACT
Regeneration of skeletal muscle upon injury is a complex process, involving activation of satellite cells, followed by migration, fusion, and regeneration of damaged myofibers. Previous work concerning the role of the mitogen activated protein (MAP) kinase signaling pathways in muscle injury comes primarily from studies using chemically induced wounding. The purpose of this study was to test the hypothesis that physical injury to skeletal muscle cells in vitro activates the MAP kinase signaling pathways. We demonstrate that extracellular signal regulated kinases (ERKs) 1, 2, and p38 are rapidly and transiently activated in response to injury in C2C12 cells, and are primarily localized to cells adjacent to the wound bed. Culture medium from wounded cells is able to stimulate activation of p38 but not ERK in unwounded cells. These results suggest that both ERK and p38 are involved in the response of muscle cells to physical injury in culture, and reflect what is seen in whole tissues in vivo.