Muscarinic Acetylcholine Receptors Activate Expression of the Egr Gene Family of Transcription Factors

Abstract

In order to search for genes that are activated by muscarinic acetylcholine receptors (mAChRs), we used an mRNA differential display approach in HEK293 cells expressing m1AChR. The zinc-finger transcription factor genes Egr-1, Egr-2, and Egr-3 were identified. Northern blot analyses confirmed that mRNA levels of Egr-1, Egr-2, and Egr-3 increased readily after m1AChR stimulation and that a maximum was attained within 50 min. At that time, Egr-4 mRNA was also detectable. Western blots and electromobility shift assays demonstrated synthesis of EGR-1 and EGR-3, as well as binding to DNA recognition sites in response to m1AChR activation. Activation of m1AChR increased transcription from EGRdependent promoters, including the acetylcholinesterase gene promoter. Activity-dependent regulation of Egr-1 mRNA expression and EGR-1 protein synthesis was also observed in cells expressing m2, m3, or m4AChR subtypes. Increased EGR-1 synthesis was mimicked by phorbol myristate acetate, but not by forskolin, and receptor-stimulated EGR-1 synthesis was partially inhibited by phorbol myristate acetate down-regulation. Together, our results demonstrate that muscarinic receptor signaling activates the EGR transcription factor family and that PKC may be involved in intracellular signaling. The data suggest that transcription of EGRdependent target genes, including the AChE gene, can be under the control of extracellular and intracellular signals coupled to muscarinic receptors. Muscarinic acetylcholine receptors (mAChRs)1 are members of a superfamily of G protein-coupled cell surface receptors with seven transmembrane domain topology. Five subtypes of mAChRs (m1-m5) can be divided according to their signaling mechanisms: m1, m3, and m5 AChRs preferentially couple to the pertussis toxin-insensitive Gq/G11 proteins that stimulate phosphoinositide hydrolysis, whereas m2 and m4 subtypes couple to the G i /G o proteins that predominantly inhibit adenylyl cyclase (1, 2). In brain, mAChRs are involved in such functions as attention, learning, memory, and cognition (3, 4). m1 and m3 AChR subtypes are localized to the somatodendritic cell surfaces of large pyramidal neurons throughout the cortex and the hippocampus, as well as on small cholinergic interneurons in the striatum. In contrast, m2 and m4 AChRs are predominantly present on axons of the large basal forebrain projection neurons that innervate cholinergic target cells throughout the cortex and the hippocampus. Activation of the postsynaptic m1/ m3/m5 AChR family by acetylcholine triggers a large variety of distinct signaling cascades including phospholipase D, adenylyl cyclase, phospholipase A2, the generation of diacylglycerol which activates protein kinase C (PKC) and couples mAChRs to the ERK-MAP-kinase signaling cascade, activation of endoplasmic reticulum IP3 receptors, stimulation of ligand-operated cell-surface Ca 2ϩ channels, and the activity of voltagegated potassium channels (5-12). Cellular responses of mAChRs include the activation of neurite outgrowth, the finetuning of me...

Morphogenetic roles of acetylcholine.

“…Other morphogenetic roles for ACh during vertebrate embryogenesis are suggested by the early presence of choline acetyltransferase (ChAT), cholinesterases, and muscarinic cholinergic receptors (mAChR) in mesenchyme and developing cartilage of the chick limbud (13,14) and myotomes (15 (18)(19)(20). Recent evidence suggests that activation of Gqcoupled M1, M3, and M5 mAChR receptor subtypes stimulates mitogen-activated protein kinase by PLC-dependent and PLC-independent mechanisms (21), and that mAChR activate the Egr family of transcription factors (22). These findings may shed light on mechanisms underlying mitogenic effects of mAChR activation.…”

Genetic analysis of the calcineurin pathway identifies members of the EGR gene family, specifically EGR3 , as potential susceptibility candidates in schizophrenia

“…It is noteworthy that expression of the entire family of EGRs can be regulated by muscarinic acetylcholine receptors (35). Mounting evidence suggests the alteration of the muscarinic cholinergic system in schizophrenia (36).…”

Muscarinic Acetylcholine Receptors Induce the Expression of the Immediate Early Growth Regulatory Gene CYR61

“…It has been shown that recombinant expressed mAChR subtypes in HEK 293 cells are coupled to the same signal transduction pathways as in neuronal cells and mediate mAChR-related processing of the amyloid precursor protein in the same manner as in central nervous system (13,14,46,59). In addition to that the expression of the immediate early transcription factor genes Egr-1/Krox24 and Egr-2/Krox20 is induced by mAChR stimulation in HEK 293 m1 cells as well as in brains from rats that were treated with the mAChR agonist pilocarpine (29,30,53).…”

“…These include immediate early transcription factors and activity-dependent genes with potential functions in modulating long term memory by structural modifications of preexisting synapses, in the generation of new neuronal connections, as well as in neurite extension and dendritic ramification (23)(24)(25)(26)(27). Cholinergic mechanisms are involved in learning and memory processes in that mAChRs promote the generation of long term potentiation (28) and activate the expression of the transcription factors c-fos, jun-B, Egr-1, Egr-2, Egr-3, and Egr-4 (29,30).…”