MUSCLE AND NEURAL ISOFORMS OF AGRIN INCREASE UTROPHIN EXPRESSION IN CULTURED MYOTUBES VIA A TRANSCRIPTIONAL REGULATORY MECHANISM

Duchenne muscular dystrophy is a prevalent X-linked neuromuscular dise ase for which there is currently no cure, Recently, it was demonstrate d in a transgenic mouse model that utrophin could functionally compens ate for the lack of dystrophin and alleviate the muscle pathology (Tin sley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I. , and Davies, K. E. (1996) Nature 384, 349-353). In this context, it t hus becomes essential to determine the cellular and molecular mechanis ms presiding over utrophin expression in attempts to overexpress the e ndogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophi n gene expression and postulated that nerve-derived trophic factors me diate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A, O., Denn is, C, L., Tinsley, J, M,, Robertson, G. S., Davies, K. E, Cartaud, J. , and Jasmin, B. J. (1997) J. Biol. Chem. 272, 8117-8120). In the pres ent study, we have therefore focused on the effect of agrin on utrophi n expression in cultured C2 myotubes. In response to Torpedo-, muscle- , or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expressi on of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were cause d by parallel increases in the transcriptional activity of the utrophi n gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24-48 h. Under these conditions, both muscle- and nerve-derived a grin increased the activity of beta-galactosidase, indicating that agr in treatment led, directly or indirectly, to the transcriptional activ ation of the utrophin gene. Furthermore, this increase in transcriptio nal activity in response to agrin resulted from a greater number of my onuclei expressing the 1.3 kilobase pair utrophin promoter-nlsLacZ con struct. Deletion of 800 base pairs 5' from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of t he utrophin promoter to exogenously applied agrin. In addition, site-d irected mutagenesis of an N-box motif contained within this 800-base p air fragment demonstrated its essential contribution in this regulator y mechanism. Finally, direct gene transfer studies performed in vivo f urther revealed the importance of this DNA element for the synapse-spe cific expression of the utrophin gene along multinucleated muscle fibe rs. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agr in is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic pr oteins. Together, these observations are therefore relevant for our ba sic understanding of the events involved in the assembly and maintenan ce of the postsynaptic membrane domain of the neuromuscular junction a nd for the potential use of utrophin as a therapeutic strategy to coun teract the effects of Duchenne muscular dystrophy.