NFATc1 activates the acetylcholinesterase promoter in rat muscle

Abstract

Nuclear factor of activated T-cells (NFAT) plays a role in the response of muscle to chronic contractile activity that can result in fiber type switching and hypertrophy. These effects are due in part to activation of target genes following Ca 2+ -mediated nuclear translocation of NFAT. Acetylcholinesterase (AChE), a component of the neuromuscular junction, is regulated by chronic muscle and nerve activity through changes in intracellular Ca 2+, suggesting that the Ache gene may be a potential downstream target of NFAT signaling. To determine whether elements of the Ache promoter are modulated by NFAT, we transiently co-expressed reporter constructs driven by fragments of the Ache promoter with an NFATc1 that is constitutively translocated to the nucleus [NFATc1(S fi A)] in rat muscle cultures. NFATc1 potentiated reporter activity when co-transfected with constructs containing Ache genomic elements from )1280 to )490 bp upstream of transcription initiation. Electrophoretic mobility shift assays demonstrated strong binding to a potential NFAT element at )793 bp and weaker binding to one at )678 bp. Co-transfection of promoter fragments, containing the binding sites at )793 and at )678 bp, with NFATc1(S fi A) potentiated reporter activity, supporting sites of interaction with NFAT. Our data suggests a role for NFAT as a modulator of Ache gene transcription. Keywords: acetylcholinesterase, muscle culture, nuclear factor of activated T-cells, promoter, transcription factor. Acetylcholinesterase (AChE, EC 3.7.1.1) is a serine hydrolase produced by skeletal muscle required for termination of neurotransmission. AChE activity is altered by physiological changes such as muscle contractile pattern, fiber type, and innervation. One common element underlying these changes is the modulation of intracellular Ca 2+ . Elevation of intracellular Ca 2+ increases Ache transcript levels during differentiation of myoblasts to myotubes (Fuentes and Taylor 1993). One pathway of Ca 2+ signaling during myogenesis is through the activation of the Ca 2+ -dependent phosphatase, calcineurin, ultimately resulting in an increase in the stability of mRNA encoding AChE (Luo et al. 1999). Ca 2+ signaling also has been implicated in the regulation of AChE during fast-to-slow fiber type switching. AChE activity is lower in slow-twitch than in fast-twitch muscle (Bacou et al. 1982;Lomo et al. 1985;Fernandez and Donoso 1988). AChE transcripts are more abundant in fast-twitch muscle but are reduced after chronic low-frequency stimulation that results in a fast-to-slow fiber type conversion (Sketelj et al. 1998). AChE transcripts are also increased in skeletal muscle hypertrophy resulting from chronic exercise (Sveistrup et al. 1995). These studies indicate that Ca 2+ -dependent signaling pathways regulate AChE in muscle and suggest that the Ache gene may be a downstream target of calcineurin signaling. One downstream target of calcineurin is NFAT (nuclear factor of activated T cells; reviewed in Rao et al. 1997 Abbreviations used: AChE, acety...

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

“…Mouse AChE promoter region contains three consensus binding sites for NFAT: one of them is conserved in mouse, rat, and human. However, the constitutively active NFATc1, instead of inhibiting, moderately stimulated the activity of the AChE gene promoter in primary rat muscle culture (Cohen and Randall, 2004). Conversely, in line with our results in adult muscles, CsA-induced inhibition of calcineurin increased the levels of AChE mRNA in cultured C2C12 myotubes, but this was primarily attributable to increased AChE mRNA stability and not to enhanced transcription (Luo et al, 1999).…”

FHL2 Binds Calcineurin and Represses Pathological Cardiac Growth

“…RCAN1.4 and BNP transcripts were analyzed as readouts, as they are direct targets of NFAT (8,43,51), and both are activated in hypertrophy (22,26). We report that increases in RCAN1.4 and BNP levels are amplified in isoproterenol-treated FHL2 Ϫ/Ϫ mice.…”

Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis

“…It has been reported that the AChE promoter contains AP-1, AP-2, GRE, binding sites (Getman et al, 1995;Perry et al, 2002;Cohen and Randall, 2004;Meshorer et al, 2004;Meshorer et al, 2005). c-Jun and c-fos can form AP-1 transcription factor.…”