Transcriptional response to muscarinic acetylcholine receptor stimulation: regulation of Egr-1 biosynthesis by ERK, Elk-1, MKP-1, and calcineurin in carbachol-stimulated human neuroblastoma cells.
Carbachol-mediated activation of type M(3) muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5'-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M(3) muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.
Rössler
Oliver G
OG
Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, Building 44, D-66421 Homburg, Saarland, Germany.
Henss
Isabell
I
Thiel
Gerald
G
D019703:Calcineurin / Q000378:metabolism*; D002217:Carbachol / Q000008:administration & dosage*; D045744:Cell Line, Tumor; D004277:DNA, Single-Stranded / Q000096:biosynthesis*; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000008:administration & dosage; D009447:Neuroblastoma / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D050807:ets-Domain Protein Elk-1 / Q000378:metabolism*
C400152:DNA enzyme ED5; D004277:DNA, Single-Stranded; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D050807:ets-Domain Protein Elk-1; D002217:Carbachol; D020928:Mitogen-Activated Protein Kinases; D019703:Calcineurin; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1