SummaryAccumulation of adipocytes and collagen type-I-producing cells (fibrosis) is observed in muscular dystrophies. The origin of these cells had been largely unknown, but recently we identified mesenchymal progenitors positive for platelet-derived growth factor receptor alpha (PDGFRa) as the origin of adipocytes in skeletal muscle. However, the origin of muscle fibrosis remains largely unknown. In this study, clonal analyses show that PDGFRa + cells also differentiate into collagen type-I-producing cells. In fact, PDGFRa + cells accumulated in fibrotic areas of the diaphragm in the mdx mouse, a model of Duchenne muscular dystrophy. Furthermore, mRNA of fibrosis markers was expressed exclusively in the PDGFRa + cell fraction in the mdx diaphragm. Importantly, TGF-b isoforms, known as potent profibrotic cytokines, induced expression of markers of fibrosis in PDGFRa + cells but not in myogenic cells. Transplantation studies revealed that fibrogenic PDGFRa + cells mainly derived from pre-existing PDGFRa + cells and that the contribution of PDGFRa 2 cells and circulating cells was limited. These results indicate that mesenchymal progenitors are the main origin of not only fat accumulation but also fibrosis in skeletal muscle.

ABSTRACT

No abstract

No abstract

Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. It is an inner nuclear membrane protein and phosphorylated in a cell cycle-dependent manner. However, the means of phosphorylation of emerin are poorly understood. We investigated the regulation mechanism for the binding of emerin to chromatin, focusing on its cell cycle-dependent phosphorylation in a Xenopus egg cell-free system. It was shown that emerin dissociates from chromatin depending on mitotic phosphorylation of the former, and this plays a critical role in the dissociation of emerin from barrier-to-autointegration factor (BAF). Then, we analyzed the mitotic phosphorylation sites of emerin. Emerin was strongly phosphorylated in an M-phase Xenopus egg cell-free system, and five phosphorylated sites, Ser 49 , Ser 66 , Thr 67 , Ser 120 , and Ser 175 , were identified on analysis of chymotryptic and tryptic emerin peptides using a phosphopeptide-concentrating system coupled with a Titansphere column, which specifically binds phosphopeptides, and tandem mass spectrometry sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at Ser 175 regulates the dissociation of emerin from BAF. The nuclear envelope (NE)2 is a highly dynamic structure that disassembles at the onset of mitosis and reassembles on the surface of chromatin during telophase in vertebrates. These changes of NE are crucial for cell cycle progression. The NE consists of an outer nuclear membrane, inner nuclear membrane, nuclear pore complex, and nuclear lamina. The inner nuclear membrane contains integral membrane proteins, i.e. lamin B receptor (LBR), lamina-associated polypeptide-2␤ (LAP2␤), emerin, MAN1, and others, which interact with DNA and/or chromatin, and these proteins are proposed to participate in nuclear membrane targeting to chromatin at an early stage of nuclear assembly (1). The interactions between some of the inner nuclear membrane proteins and chromatin are regulated through phosphorylation of these inner nuclear membrane proteins. The phosphorylation mechanisms for LBR and LAP2␣ and 2␤ are well understood (2-7). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3). LAP2␤ binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2␤ cancels this binding (4). Phosphorylation of these inner nuclear proteins, therefore, is likely to be one of the key mechanisms that control the interactions between the inner nuclear proteins and components of the nuclear lamina as well as chromatin. In this study, we focused on the mitotic phosphorylation of emerin, one of the inner nuclear membrane proteins.Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8) and is phosphorylated in a cell cycle-dependent manner (9). Emerin belongs to the LEM (LAP2␤, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10). These ...