Calcineurin Enhances mRNA Stability of Acetylcholinesterase During Muscle Differentiation in C2-C12 Cells

Abstract

Differentiation from myoblasts to myotubes in mouse C2-C12 cell line results in increased expression of acetylcholinesterase (AChE), primarily due to stabilization of the mRNA (Fuentes and Taylor, Neuron 1993). Regulation of intracellular calcium plays an important role in the message stabilization process (Luo et al., JBC 1994). In a search for the calcium-sensitive pathway(s) responsible for AChE mRNA stabilization, we found that treatment of differentiating C2-C12 muscle cells with cyclosporine A (CsA), an immunosuppressive agent that inhibits calcium/calmodulin dependent phosphatase calcineurin after its binding to intracellular cyclophilin A, resulted in dose- and time-dependent increases of AChE mRNA and protein levels. The increased expression appeared primarily due to increased stability of the AChE mRNA because transcriptional rate of the gene did not change after the treatment. The CsA action is specific and not due to alteration of muscle differentiation because treated cells displayed normal muscle cell morphology and expression of nicotinic acetylcholine receptors (nAChR). Treatment with inactive analog of CsA, cyclosporine H, or another immunosuppressant, FK506, neither of which binds to cyclophilin A, was without effect. In addition, the CsA action is associated with muscle differentiation, rather than an early event of differentiation triggered by CsA treatment, because CsA treated cells displayed normal rate of AChE expression in CsA free medium during further differentiation. Furthermore, the CsA action was not due to arresting the cells in G0 phase because treatment with CsA before induction of terminal differentiation did not increase AChE expression. In an effort to identify the cellular mediator of the CsA action, we round that expression levels of cyclophilin A were similar in myoblasts and myotubes.