G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities.

Carbamylated erythropoietin does not alleviate signs of dystrophy in mdx mice.

Erythropoietin promotes myoblast proliferation and inhibits fibrosis and thus it could impede the pathogenesis of muscle degenerative diseases. However, its stimulation of erythropoiesis limits its use as a therapeutic agent. An erythropoietin analog, carbamylated erythropoietin (C-EPO), retains these protective actions, yet it does not interact with the erythropoietin receptor. To determine whether treatment with C-EPO alleviates the signs of muscular dystrophy in an animal model of Duchenne muscular dystrophy, we treated mdx mice with intraperitoneal injections of 50 µg/kg and 100 µg/kg C-EPO for 4 and 12 weeks, and we monitored weight, serum creatine kinase levels, and changes in muscle histology. Moderate histological improvement was observed at 4 weeks, which did not translate into a significantly decreased level of serum creatine kinase. At the doses tested, C-EPO is not an effective therapeutic for the treatment of a mouse model of Duchenne muscular dystrophy.

Requirement of the tissue-restricted homeodomain transcription factor Nkx6.3 in differentiation of gastrin-producing G cells in the stomach antrum.

Many homeodomain transcription factors function in organogenesis and cell differentiation. The Nkx family illustrates these functions especially well, and the Nkx6 subfamily controls differentiation in the central nervous system and pancreas. Nkx6.3, a recent addition to this subfamily, overlaps Nkx6.1 and Nkx6.2 in expression in the hindbrain and stomach. Nkx6.3 transcripts localize in the epithelium of the most distal stomach region, the antrum and pylorus; expression in the adult intestine is lower and confined to the proximal duodenum. Nkx6.3(-)(/)(-) mice develop and grow normally, with a grossly intact stomach and duodenum. These mice show markedly reduced gastrin mRNA, many fewer gastrin-producing (G) cells in the stomach antrum, hypogastrinemia, and increased stomach luminal pH, with a corresponding increase in somatostatin mRNA levels and antral somatostatin-producing (D) cells. They express normal levels of other transcription factors required for gastric endocrine cell differentiation, Pdx1, Pax6, and Ngn3; conversely, Ngn3(-)(/)(-) mice, which also show reduced gastrin levels, express Nkx6.3 normally. These studies implicate Nkx6.3 as a selective regulator of G- and D-cell lineages, which are believed to derive from a common progenitor, and suggest that it operates in parallel with Ngn3.

C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia.

We have identified a gene by microarray analysis that is located on chromosome 6 (c6orf32), whose expression is increased during human fetal myoblast differentiation. The protein encoded by c6orf32 is expressed both in myogenic and non-myogenic primary cells isolated from 18-week old human fetal skeletal muscle. Immunofluorescent staining indicated that C6ORF32 localizes to the cellular cytoskeleton and filopodia, and often displays polarized expression within the cell. mRNA knockdown experiments in the C2C12 murine myoblast cell line demonstrated that cells lacking c6orf32 exhibit a myogenic differentiation defect, characterized by a decrease in the expression of myogenin and myosin heavy chain (MHC) proteins, whereas MyoD1 was unaltered. In contrast, overexpression of c6orf32 in C2C12 or HEK293 cells (a non-muscle cell line) promoted formation of long membrane protrusions (filopodia). Analysis of serial deletion mutants demonstrated that amino acids 55-113 of C6ORF32 are likely involved in filopodia formation. These results indicate that C6ORF32 is a novel protein likely to play multiple functions, including promoting myogenic cell differentiation, cytoskeletal rearrangement and filopodia formation.