Muscular activity regulates the expression of ColQ subunit of acetylcholinesterase : a signaling pathway mediated by Ca²⁺/ calmodulin-dependent protein kinase II

At vertebrate neuromuscular junction (nmj), the asymmetric acetylcholinesterase (AChE) is highly concentrated and is anchored to the synaptic basal lamina through the presence of a collagen-tailed subunit (ColQ). There are two ColQ mRNAs being expressed, namely ColQ-1 and ColQ-1a, which are driven by two distinct promoters. ColQ-1 mRNA is mainly expressed in slow muscle throughout the entire muscle fiber while ColQ-1a mRNA is preferentially expressed in fast muscle with a synaptic-specific manner. Recently, a calcitonin gene-related peptide (CGRP)-induced cAMP-dependent signaling pathway was identified to direct the synaptic expression of ColQ-1a. However, the molecular mechanism in regulating the extra-synaptic expression of ColQ is still unknown. The nerve-evoked muscular activity was found to regulate the expression of acetylcholine receptor (AChR) and AChE in the extra-synaptic region in muscles. This led us to speculate the role of muscular activity in regulating ColQ gene expression. To study the regulatory role of muscular activity on ColQ at the nmj, mouse C2C12 muscle cells were used as a model system. The expression levels of ColQ-1 and ColQ-1a mRNAs were determined by real-time polymerase chain reaction (PCR). Acetylcholine and nicotine, both are AChR agonists, were used to mimic the muscular activity. After the drug treatments, the expression of ColQ-1 and ColQ-1a mRNAs were markedly increased; this induction was abolished by the pre-treatment of either AChR blocker (α-bungarotoxin) or Ca2+ chelator (BAPTA-AM). In order to investigate the signaling pathway for mediating the muscular activity-induced ColQ expression, we targeted a well-known downstream effector of muscular activity, Ca2+/ calmodulin-dependent protein kinase II (CaMKII). Acetylcholine-mediated muscular activity activated CaMKII phosphorylation in a time-dependent manner. In line with the above findings, the over expression of CaMKII enhanced both ColQ-1 and ColQ-1a mRNA levels in cultured myotubes, and the augmentation could be inhibited by the pretreatment of a specific CaMKII blocker, KN62. Several lines of evidence indicated that myocyte enhancer factor-2 (MEF2) is a potential downstream substrate of CaMKII and is also involved in regulating numerous muscle-specific genes. Unexpectedly, over expression of MEF2C alone could not cause a significant increase of ColQ-1, or ColQ-1a, mRNA in C2C12 myotubes. It might due to the presence of histone deacetylase 4 (HDAC4) in the nucleus to repress the activity of MEF2C. In contrast, the co-expression of MEF2C with CaMKII in cultured myotubes could potentiate the CaMKII-induced ColQ expression. We speculated that the activation of CaMKII could phosphorylate HDAC4 and cause the release of MEF2C from HDAC4-MEF2C complex. Hence, the free MEF2C could then bind to the ColQ promoters and trigger the gene transcriptions of ColQ-1 and ColQ-1a. In summary, the results reveal a detailed molecular mechanism of acetylcholine-mediated muscular activity to up-regulate ColQ expression in muscles via a CaMKII-dependent signaling pathway. CaMKII indirectly activates the transcription factor, MEF2C, by causing the dissociation of MEF2C from the repressor complex. Moreover, these findings explain the extra-synaptic expression of ColQ in muscle that is important for the localization of asymmetric AChE at the nmjs in order to prevent the acetylcholine leakage from the synaptic region