ABSTRACT
Activator protein-1 (AP-1) is a ubiquitous transcription factor that paradoxically also has some tissue-specific functions. In skeletal muscle cells, we document that the AP-1 subunit, Fra-2, is expressed in the resident stem cells (Pax7-positive satellite cells) and also in the analogous undifferentiated 'reserve' cell population in myogenic cultures, but not in differentiated myofiber nuclei. Silencing of Fra-2 expression enhances the expression of differentiation markers such as muscle creatine kinase and myosin heavy chain, indicating a possible role of Fra-2 in undifferentiated myogenic progenitor cells. We observed that Fra-2 is a target of cytokine-mediated extracellular signal-regulated kinase-1/2 signaling in cultured muscle cells, and extensive mass spectrometry and mutational analysis identified S320 and T322 as regulators of Fra-2 protein stability. Interestingly, Fra-2 S320 phosphorylation occurs transiently in activated satellite cells and is extinguished in myogenin-positive differentiating cells. Thus, cytokine-mediated Fra-2 expression and stabilization is linked to regulation of myogenic progenitor cells having implications for the molecular regulation of adult muscle stem cells and skeletal muscle regeneration.
Subject(s)
Fos-Related Antigen-2/metabolism , MAP Kinase Signaling System , Satellite Cells, Skeletal Muscle/physiology , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Differentiation , Cell Line , Cytokines/physiology , Fos-Related Antigen-2/chemistry , Mice , Molecular Sequence Data , Muscle Development , Mutagenesis, Site-Directed , Phosphorylation , Protein Processing, Post-Translational , Protein StabilityABSTRACT
CD11c, a member of the ß(2) integrin family of adhesion molecule, is expressed on the surface of myeloid lineages and activated lymphoid cells and forms a heterodimeric receptor with CD18. We analyzed the mouse CD11c promoter structure to elucidate the transcriptional regulation in dendritic cells (DCs). By reporter assay, the -84/-65 region was identified to be essential for activity of the mouse CD11c promoter in the mouse bone marrow-derived (BM) DCs and monocyte cell line RAW264.7. An electrophoretic mobility shift assay using a number of antibodies against transcription factors revealed that the target region was recognized by a complex including JunD and Fra2, which are transcription factors belonging to the AP-1 family. The direct interaction of JunD and Fra2 with the CD11c promoter was further confirmed by a chromatin immunoprecipitation assay using CD11c-positive cells purified from BMDCs. Finally, mouse JunD and/or Fra2 siRNA was introduced into BMDCs to evaluate the involvement of these factors against CD11c transcription and found that Fra2 siRNA reduced cell surface expression level of CD11c. These results indicate that AP-1 composed with JunD and Fra2 protein plays a primary role in enhancing the transcription level of the CD11c gene in DC.
