Myocyte enhancer factor 2 mediates acetylcholine-induced expression of acetylcholinesterase-associated collagen ColQ in cultured myotubes

Abstract

The collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) in vertebrate muscles. Two ColQ transcripts, ColQ-1 and ColQ-1a, driven by two distinct promoters are expressed differentially in mammalian slow- and fast-twitch muscles, respectively. Such expression patterns are determined by the contractile activity in different muscle fiber types. To reveal the regulatory role of muscular activity on ColQ expression, acetylcholine and nicotine were applied onto C2C12 muscle cells: the challenge increased the expression of ColQ-1/ColQ-1a mRNAs. The agonist challenge induced the phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). In parallel, over expression of an active mutant of CaMKII enhanced both ColQ-1/ColQ-1a mRNA levels in cultured C2C12 myotubes. Moreover, the over expression of myocyte enhancer factor 2 (MEF2), a downstream mediator of CaMKII, in the myotubes potentiated the CaMKII-induced ColQ expression. The current results reveal a signaling cascade that drives the expression profiles of ColQ in responding to activity challenge in cultured myotubes.

Introduction

In vertebrate neuromuscular junctions (nmjs), acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme that is concentrated at the synaptic basal lamina and the post-synaptic muscle fiber (Massoulié et al., 2005). A single ACHE gene produces several types of catalytic subunits by alternative splicing: a single splice variant called type T (AChE_T) is expressed in adult mammalian muscles. AChE_T catalytic subunits produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, collagenous protein (ColQ) and proline-rich membrane anchor (PRiMA), which allow their functional localization in synapses (Massoulié et al., 2005). Collagenous ColQ characterizes the collagen-tailed forms (A forms) of AChE (Tsim et al., 1988, Krejci et al., 1991), which are localized at the basal lamina of vertebrates nmjs (Peng et al., 1999, Cartaud et al., 2004); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The ColQ-associated AChE is the predominant enzyme at the nmjs.

At nmjs, the motor nerve provides two distinct mechanisms to regulate expression and/or distribution of AChE forms: release of trophic factor(s) and nerve-evoked electrical activity (Haynes et al., 1986, Choi et al., 2001., Rossi et al., 2003, Pregelj et al., 2007). This notion is strongly supported by different lines of evidence. The appearance of ColQ-associated AChE coincides with the establishment of nerve–muscle contacts (Sketelj and Brzin, 1985, Deprez et al., 2003), and its expression level decreases dramatically after the nerve denervation (Fernandez and Stiles, 1984). A nerve-derived trophic factor, calcitonin gene-related peptide (CGRP), directs the production of AChE_T transcripts (Choi et al., 1998, Boudreau-Larivière and Jasmin, 1999, Rossi et al., 2003) and ColQ (Choi et al., 2007); even though contrary results were revealed in CGRP knock out mice that the nmjs of these mice were normal (Lu et al., 1999). In addition, fast-twitch- and slow-twitch muscles have distinct patterns of AChE forms (Sketelj and Brzin, 1985, Gisiger and Stephens, 1988, Sketelj et al., 1998, Boudreau-Larivière et al., 2000). In fast-twitch muscle, the A12 form is largely predominant and exclusively localized at the synapses, while in slow-twitch muscle, the A8 and A4 forms are relatively abundant and found in synaptic and extra-synaptic regions of muscle fibers (Sketelj and Brzin, 1985, Cresnar et al., 1994, Massoulié et al., 2005). These differences could be explained by the spatial expressions of AChE_T and ColQ transcripts: ColQ is expressed at the nmjs of fast-twitch muscles, but it is uniformly expressed in the entire slow-twitch muscle fiber (Krejci et al., 1999). Thus, the formation of different forms of AChE in muscle could depend on the abundance of AChE_T and ColQ; however, the signals triggering the transcript expressions of these two genes are not known.

In human and rat, the COLQ gene (∼ 50 kb in length) possesses two origins of transcription located ∼ 23 kb apart, generating alternative exons 1 and 1a (Donger et al., 1998, Ohno et al., 1998), producing 2 transcripts, ColQ-1 and ColQ-1a, that are expressed in slow-twitch and fast-twitch muscles, respectively (Lee et al., 2004, Choi et al., 2007). Exon 1 and exon 1a encode essentially the signal peptides, and mature ColQ-1 and ColQ-1a subunits possess nearly identical sequences (Krejci et al., 1997). The robust difference of ColQ expression in fast-twitch and slow-twitch muscles strongly suggests that muscular activity could play a critical role in regulating the gene expression; however, the signals that trigger a high ColQ expression in slow-twitch muscle but not in fast-twitch muscle are still not known. As to mimic muscular activity in an in vitro system, we employed acetylcholine receptor (AChR) agonists in cultured C2C12 myotubes to study the expression pattern of ColQ-1 and ColQ-1a: C2C12 muscle cell line expresses both ColQ transcripts and produces both types of muscle fibers in culture (Siow et al., 2002, Lee et al., 2004, Choi et al., 2007). Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and its downstream mediator myocyte enhancer factor 2 (MEF2) are shown here to be key mediators in directing acetylcholine (ACh)-induced ColQ expression.