Abstract
The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Base Sequence
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Binding Sites
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Blotting, Western
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COS Cells
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Calpain / genetics*
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Calpain / metabolism
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Cells, Cultured
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Chlorocebus aethiops
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DNA Primers / chemistry
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DNA-Binding Proteins / physiology*
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Fibroblasts / physiology
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Gene Expression Regulation*
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Humans
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Immunoenzyme Techniques
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Luciferases / metabolism
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MEF2 Transcription Factors
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Mice
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Molecular Sequence Data
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Muscle Development / physiology*
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Muscle Proteins / antagonists & inhibitors
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Muscle Proteins / genetics
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Muscle Proteins / pharmacology
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MyoD Protein / antagonists & inhibitors
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MyoD Protein / genetics*
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MyoD Protein / pharmacology
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Myoblasts / physiology*
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Myogenic Regulatory Factor 5
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Myogenic Regulatory Factors
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Myogenin / antagonists & inhibitors
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Myogenin / genetics*
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Myogenin / pharmacology
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Oligonucleotides, Antisense / pharmacology
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Promoter Regions, Genetic*
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Recombinant Proteins
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Trans-Activators*
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Transcription Factors / metabolism
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Transcription Factors / physiology*
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Transcriptional Activation / genetics*
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Transfection
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Up-Regulation
Substances
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DNA Primers
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DNA-Binding Proteins
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MEF2 Transcription Factors
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MYF5 protein, human
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MYOG protein, human
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Muscle Proteins
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Myf5 protein, mouse
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MyoD Protein
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Myog protein, mouse
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Myogenic Regulatory Factor 5
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Myogenic Regulatory Factors
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Myogenin
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Oligonucleotides, Antisense
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Recombinant Proteins
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Trans-Activators
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Transcription Factors
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Luciferases
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Calpain
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m-calpain