Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion
Deakin University, AustraliaJournal of Biological Chemistry (Impact Factor: 4.57). 12/2012; 288(3). DOI: 10.1074/jbc.M112.429647
Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Since the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesised that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15 or fulllength V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia and muscular dystrophy.
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ABSTRACT: Aim: We investigated the effect of the number of cytosine-adenine (CA) repeats in the ADAMTS9 promoter region on breast cancer lymphatic metastasis. Materials and methods: Thirty-one postoperative breast cancer patients were selected and examined retrospectively. The patients were classified into 2 groups: metastatic or nonmetastatic. Thirty healthy women were selected as the control group, and their peripheral blood was obtained. Following DNA isolation from the cancer tissue specimens and peripheral blood, the promoter region of the ADAMTS9 gene was directly sequenced and the number of CA repeats was determined. Results: The number of CA repeats ranged between 19 and 21 in the control and metastatic groups. However, in the nonmetastatic group, the number of CA repeats ranged between 17 and 18. This difference in the median number of CA repeats between the control group and the nonmetastatic group was statistically significant. Conclusion: A potential relationship may exist between lymphatic metastasis in breast cancer and the number of CA repeats in the promoter region of the ADAMTS9 gene. Our study indicates a potential association between the number of CA microsatellite repeats in the promoter region of the ADAMTS9 gene and breast cancer lymphatic metastasis.
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ABSTRACT: SUMMARY Exogenous hyaluronan is known to alter muscle precursor cell proliferation, migration and differentiation, ultimately inhibiting myogenesis in vitro. The aim of the current study was to investigate the role of endogenous hyaluronan synthesis during myogenesis. In quantitative PCR studies, the genes responsible for synthesising hyaluronan were found to be differentially regulated during muscle growth, repair and pathology. Whilst all Has genes (Has1, Has2 and Has3) were differentially regulated in these models, only Has2 gene expression consistently associated with myogenic differentiation. During myogenic differentiation in vitro, Has2 was the most highly expressed of the synthases and increased after induction of differentiation. To test whether this association between Has2 expression and myogenesis relates to a role for Has2 in myoblast differentiation and fusion, C2C12 myoblasts were depleted of Has2 by siRNA and induced to differentiate. Depletion of Has2 inhibited differentiation and caused a loss of cell associated hyaluronan and the hyaluronan dependent pericellular matrix. The inhibition of differentiation caused by loss of hyaluronan was confirmed with the hyaluronan synthesis inhibitor 4- methylumbelliferone. In hyaluronan synthesis blocked cultures, restoration of the pericellular matrix could be achieved through the addition of exogenous hyaluronan and the proteoglycan versican, but was not sufficient to restore differentiation to control levels. These data indicate that intrinsic hyaluronan synthesis is necessary for myoblasts to differentiate and form syncytial muscle cells, but the hyaluronan dependent pericellular matrix is not sufficient to support differentiation alone; additional hyaluronan dependent cell functions that are yet unknown may be required for myogenic differentiation.
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ABSTRACT: The proteoglycanase clade of the ADAMTS superfamily shows preferred proteolytic activity toward the hyalectan/lectican proteoglycans as follows: aggrecan, brevican, neurocan, and versican. ADAMTS15, a member of this clade, was recently identified as a putative tumor suppressor gene in colorectal and breast cancer. However, its biosynthesis, substrate specificity, and tissue expression are poorly described. Therefore, we undertook a detailed study of this proteinase and its expression. We report propeptide processing of the ADAMTS15 zymogen by furin activity, identifying RAKR212↓ as a major furin cleavage site within the prodomain. ADAMTS15 was localized on the cell surface, activated extracellularly, and required propeptide processing before cleaving V1 versican at position 441E↓A442. In the mouse embryo, Adamts15 was expressed in the developing heart at E10.5 and E11.5 days post-coitum and in the musculoskeletal system from E13.5 to E15.5 days post-coitum, where it was co-localized with hyaluronan. Adamts15 was also highly expressed in several structures within the adult mouse colon. Our findings show overlapping sites of Adamts15 expression with other members of ADAMTS proteoglycanases during embryonic development, suggesting possible cooperative roles during embryogenesis, consistent with other ADAMTS proteoglycanase combinatorial knock-out mouse models. Collectively, these data suggest a role for ADAMTS15 in a wide range of biological processes that are potentially mediated through the processing of versican.
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