Résumé :
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Communication n° 421. Introduction : We have previously shown that slow/oxidative muscle fibers contain significantly more utrophin in comparison to fast/glycolytic fibers (Gramolini et al., 2001). More recent studies have implicated calcineurin/NFAT signaling in directly stimulating utrophin A expression in slow fibers (Chakkalakal et al., 2003). Additionally, we have shown that over-expression of activated calcineurin in mdx mouse muscle results in increased utrophin A expression, restoration of the dystrophin complex and an attenuation of the dystrophic phenotype (Chakkalakal et al., 2004). Collectively, these findings demonstrate the importance of further defining the molecular events involved in the calcineurin-induced regulation of utrophin A expression. Objectives : Based on our previous data also showing a role for the 3'UTR in mediating differences in utrophin expression between fast and slow muscles, we examined in the present study the contribution of calcineurin signaling in this post-transcriptional regulatory cascade. Methods : Reporter Assays of utrophin 3'UTR regions were conducted by RT-PCR analysis of transfected C2C12 cells and transduced soleus and edl muscles. UV-crosslinking and RNA gel shift assays were carried out using labeled RNA probes and skeletal muscle protein extract. Results : Direct plasmid injection of skeletal muscles and transfection of C2C12 cells with reporter constructs containing regions of the utrophin 3'UTR, identified a calcineurin-responsive element capable of conferring differences in the expression of the reporter constructs between fast and slow muscles. UV-crosslinking and RNA gel shift assays using this region revealed that proteins from skeletal muscle and ranging in size from 25-50 kD, are capable of interacting with this minimal sequence. Conclusions : The identification of this calcineurin-responsive cis-element and the further characterization of interacting trans-acting factors may prove useful in fully defining the role of calcineurin in regulating utrophin in muscle and for designing pharmacological therapies based on upregulating utrophin in dystrophic muscle.
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