title: Prolactin regulation of pim-1 expression: positive and negative promoter elements.
abstract: The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to gamma-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2-11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5'-pim-1 promoter and a series of deletion mutants were ligated upstream of the chloramphenicol acetylase transferase (CAT) gene in an expression vector that was introduced into FDC/Nb2 cells, a premyeloid line that stably expresses the intermediate form of the PRLR. Analysis of PRL-treated cultures indicated that two elements [distal (DE), -427 to -336 bp and proximal (PE), - 104 to -1] but not several GAS or GAS-like sequences were required for hormone activation of the pim-1 promoter. Moreover, treatment of Nb2-11 cells with PRL activated protein binding to these elements assessed by gel mobility shift assay. Deoxyribonuclease I (DNase I) protection experiments revealed a motif containing a nuclear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when exposed to extracts from PRL-treated cells but protected by proteins from unstimulated cells. Gel mobility shift analysis of this sequence showed decreased protein binding after PRL stimulation. It is concluded that the PRLR initiates pim-1 transcription by a mechanism that involves transcriptional activation by factors that stimulate the DE- and PE-sites and derepress a NF-1-containing element. Moreover, this mechanism appears to be independent of an interaction between Stat transcription factors and GAS-like elements present within the promoter.
authors.affiliation: Department of Pharmacology & Toxicology, University of North Dakota School of Medicine and Health Sciences, Grand Forks 58202, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Borg
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.forename: D
authors.initials: D
authors.lastname: Hegge
authors.forename: R L
authors.initials: RL
authors.lastname: Stephen
authors.forename: D J
authors.initials: DJ
authors.lastname: Buckley
authors.forename: N S
authors.initials: NS
authors.lastname: Magnuson
authors.forename: A R
authors.initials: AR
authors.lastname: Buckley
mesh_terms: D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects*; D008223:Lymphoma; D016296:Mutagenesis; D051839:NFI Transcription Factors; D011388:Prolactin / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051573:Proto-Oncogene Proteins c-pim-1; D051381:Rats; D011981:Receptors, Prolactin / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D051840:Y-Box-Binding Protein 1
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D011518:Proto-Oncogene Proteins; D011981:Receptors, Prolactin; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C486169:Ybx1 protein, rat; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; C496637:Pim1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1; D003850:Deoxyribonuclease I