title: Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.
abstract: The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.
authors.affiliation: Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, Homburg, Germany.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Müller
authors.forename: Oliver G
authors.initials: OG
authors.lastname: Rössler
authors.forename: Gerald
authors.initials: G
authors.lastname: Thiel
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D016764:Cell Polarity / Q000187:drug effects / Q000235:genetics; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016350:Leucine Zippers / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011284:Pregnenolone / Q000494:pharmacology*; D061251:Primary Cell Culture; D051381:Rats; D050053:TRPM Cation Channels / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: D015220:Calcium Channels; D050053:TRPM Cation Channels; C533867:TRPM3 protein, rat; C018370:pregnenolone sulfate; D011284:Pregnenolone