title: A calcium-initiated signaling pathway propagated through calcineurin and cAMP response element-binding protein activates proenkephalin gene transcription after depolarization.
abstract: Essential components of a signal-transduction pathway regulating activity-dependent neuropeptide gene transcription have been identified. Proenkephalin (PEnk) gene activation after depolarization of chromaffin cells with 40 mM KCl was blocked by the voltage-sensitive calcium-channel blocker methoxyverapamil (D600) (30 microM) and by calcineurin inhibition with 100 nM cyclosporin A or ascomycin but not by inhibiting new protein synthesis with 0.5 microg/ml cycloheximide. KCl-induced elevation of PEnk mRNA was distinct from activation of the PEnk gene by either cAMP or protein kinase C. Twenty-five micromolar forskolin- and 100 nM phorbol 12-myristate 13-acetate-induced elevations of PEnk mRNA were cycloheximide-sensitive and were not blocked by cyclosporin A or ascomycin. KCl stimulated Ser-133 phosphorylation of cAMP response element-binding protein (CREB) in chromaffin cells, and CREB phosphorylation was blocked by both ascomycin and D600. A reporter gene containing 193 bases of the PEnk gene 5' flank driving luciferase gene expression (pENK12-Luc) transfected into chromaffin cells was transcriptionally activated by KCl depolarization. Activation was blocked by both ascomycin and D600 and required an intact CREB binding site (ENKCRE2). An oligonucleotide containing the PEnk cAMP response element-2 was gel-shifted by both unstimulated and potassium-stimulated chromaffin cell nuclear extracts into a prominent complex supershifted by CREB antibodies. Finally, stimulation of transcription of the pENK12-Luc reporter by KCl in chromaffin cells was blocked by coexpression of the CREB antagonist A-CREB but not by the AP-1 antagonist A-Fos. Stimulus-transcription coupling after depolarization in chromaffin cells occurs via calcineurin-dependent activation of CREB, a pathway distinct from cAMP- or protein kinase C-initiated signaling and independent of immediate early gene regulation.
authors.affiliation: Building 36, Room 2A-11, National Institute of Mental Health, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. eiden@codon.nih.gov
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Hahm
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.forename: Charles
authors.initials: C
authors.lastname: Vinson
authors.forename: Lee E
authors.initials: LE
authors.lastname: Eiden
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004745:Enkephalins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D015533:Transcriptional Activation
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D011189:Potassium Chloride; D019703:Calcineurin