Skeletal muscle stem cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. The JAK-STAT pathways are well characterized for their essential roles in hematopoiesis and antimicrobial immune response. Previous work in our laboratory showed that the JAK1-STAT1-STAT3 pathway plays a very important role in myogenesis in the cell culture model, but the detailed molecular mechanism is yet to be fully elucidated. In this study, we used microarray to systematically identify Jak1-STAT1 target genes during muscle cell proliferation and differentiation. Among candidate target genes identified, we chose D...[ Read more ]

Skeletal muscle stem cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. The JAK-STAT pathways are well characterized for their essential roles in hematopoiesis and antimicrobial immune response. Previous work in our laboratory showed that the JAK1-STAT1-STAT3 pathway plays a very important role in myogenesis in the cell culture model, but the detailed molecular mechanism is yet to be fully elucidated. In this study, we used microarray to systematically identify Jak1-STAT1 target genes during muscle cell proliferation and differentiation. Among candidate target genes identified, we chose Dusp27 for further detailed functional analysis. Dusp27 belongs to the dual-specificity phosphatase (DUSPs) family due to the presence of a conserved DUSP domain, and appears to be catalytically inactive because of the substitution of a conserved cystenine to serine. So far, Dusp27 has not been functionally characterized in any cell type. Interestingly, Dusp27 is preferentially expressed in the heart and skeletal muscles. Knockdown of Dusp27 in C2C12 cells consistently inhibited myogenic differentiation while overexpression of a siRNA-resistant form of Dusp27 could rescue the inhibitory effect of the siRNAs. Our data also showed that the expression levels of MyoD and MEF2 were not affected by Dusp27-siRNAs. However, the transcriptional activity of MyoD was inhibited by knockdown of Dusp27. Meanwhile, knockdown of Dusp27 leads to significant up-regulation of Cyclin D1 without affecting the cell proliferation. In vivo study also shows that Dusp27 plays a role in cardiotoxin-induced muscle regeneration. Further studies are needed to elucidate the underlying molecular mechanism.

Dusp27 is highly conserved among vertebrates including zebrafish with 47% amino-acid identity between mouse Dusp27 and its zebrafish counterpart. In order to quickly assess the role of Dusp27 in vivo, we decided to employ zebrafish as a model, as by 24-hour post-fertilization, zebrafish muscles are already fully formed. Knockdown of Dusp27 by morpholino oligonucleotides (MOs) resulted in malformation of both fast and slow muscle fibers. The specificity of MOs was confirmed by co-injection of Dusp27-GFP expressing plasmids. Over-expression of Dusp27 MO-resistant form of mRNA could rescue the effect of Dusp27 MO. In situ hybridization of several myofibril genes, including myosin heavy polypeptide 1 (Myhz1), myosin light polypeptide 2 (Mylz2), slow myosin heavy polypeptide 1 (Smyhc1), actnin 1(Acta1) and tropomyosin (Tpma), showed that knockdown of Dusp27 did not affect the expression levels of these genes. However, the myofibril formation is impaired in Dusp27 MO injected embryos. Preliminary data showed that Dusp27 may regulate myofibrillogenesis through interaction with some z-disc localized proteins, such as Tcap, CapZ, Myotilin and ZASP. Further experiments are ongoing to confirm their interaction and to uncover the mechanism by which Dusp27 regulates myofibrillogenesis. Moreover, to understand the in vivo function of Dusp27, we aimed to generate Dusp27 knockout mice. We have obtained mice with two LoxP sites flanking the exons 3-5 of the Dusp27 gene. These mice are being crossed with Pax7-Cre mice and EIIA-Cre mice to generate muscle satellite cell-specific Dusp27 knockout mice and total Dusp27 knockout mice, respectively.