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.
Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, Building 44, D-66421 Homburg, Saarland, Germany.
Oliver G
OG
Rössler
Isabell
I
Henss
Gerald
G
Thiel
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