The regulation of acetylcholinesterase by cis‐elements within intron I in cultured contracting myotubes

Abstract

The onset of spontaneous contraction in rat primary muscle cultures coincides with an increase in acetylcholinesterase (AChE) activity. In order to establish whether contractile activity modulates the rate of AChE transcript synthesis, and what elements of the gene are determinant, we examined the promoter and intron I in contracting muscle cultures. Ache genomic fragments attached to a luciferase reporter were transfected into muscle cultures that were either electrically stimulated or paralyzed with tetrodotoxin to enhance or inhibit contractions, respectively. Cultures transfected with intron I-containing constructs showed a 2-fold increase in luciferase activity following electrical stimulation, compared to tetrodotoxin treatment, suggesting that this region contains elements responding to contractile activity. Deleting a 780 bp distal region within intron I, containing an N-box element at +890 bp, or introducing a 2-bp mutation within its core sequence, eliminated the contraction-induced response. In contrast, mutating an N-box element at +822 bp had no effect on the response. Furthermore, co-transfecting a dominant negative GA-binding protein (GABP), a transcription factor known to selectively bind N-box elements, reduced the stimulation-mediated increase. Our results suggest that the N-box within intron I at +890 bp is a regulatory element important in the transcriptional response of Ache to contractile activity in muscle.

Microarray transfection analysis of conserved genomic sequences from three immediate early genes

“…A 374 bp CNS within the second intron of the human nestin gene has been demonstrated to be responsible neural progenitor cell expression during development [25; 26]. Within the first intron of the mouse acetylcholinesterase gene, a CNS containing N-box elements which bind the transcription factor GABP mediates the enhancement of acetylcholinesterase expression following muscle contraction [27]. Two CNSs within the second intron of the human BRCA1 gene contribute to transcription of that gene and the effects of these CNSs are promoter specific [28], similar to the specificity shown for the c-fos I1 sequence.…”

The Role of Muscle Activation Pattern and Calcineurin in Acetylcholinesterase Regulation in Rat Skeletal Muscles

“…However, chronic low-frequency tonic stimulation of the fast EDL muscles decreased junctional AChE activity to the low levels observed in the junctions of the slow soleus muscle (Sketelj et al, 1998). Therefore, activated calcineurin signaling mechanism might also downregulate junctional AChE expression, acting in parallel to other mechanisms regulating junctional AChE expression (Chan et al, 1999;Rossi et al, 2003;Tung et al, 2004;Cohen and Randall, 2006). The same was demonstrated for utrophin gene expression at the neuromuscular junction (Angus et al, 2005).…”

In Vitro Innervation as an Experimental Model to Study the Expression and Functions of Acetylcholinesterase and Agrin in Human Skeletal Muscle

“…Conversely, cultured primary chick [ 51 , 52 , 53 , 54 ], quail [ 55 , 56 ], rat [ 57 , 58 , 59 , 60 , 61 , 62 ] and mouse [ 34 , 63 , 64 ] skeletal muscle cells, which are often obtained from embryos or neonatal animals, display more advanced level of differentiation and contract spontaneously in the absence of innervation or electrical stimulation. Indeed, to study quiescent primary rat or mouse skeletal muscle cells tetrodotoxin (TTX), a blocker of voltage-gated Na + channels, is used to suppress spontaneous contractions [ 34 , 60 , 61 , 62 , 65 , 66 , 67 ]. Spontaneous discharge of action potentials provides one explanation for autonomous contractile activity of aneurally cultured skeletal muscle cells [ 51 ].…”

“…Spontaneous contractile activity, as well as the presence of nAChR clusters in animal myotubes, has been successfully exploited to examine effects of electromechanical activity on expression and distribution of AChE and nAChR. Indeed, by using animal embryonic cells it was possible to study effects of TTX-induced quiescence as well as spontaneous and/or electrically-stimulated muscle contractile activity on the expression of AChE [ 60 , 61 , 65 ] and nAChR [ 51 , 57 , 62 , 69 , 70 ]. For instance, expression of the 16S (A12) AChE, the major NMJ AChE species [ 5 , 71 ], is markedly increased by spontaneous contractile activity of rat [ 60 , 61 ], mouse [ 67 ] and quail [ 56 ] myotubes.…”