Prolactin regulation of pim-1 expression: Positive and negative promoter elements

The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PR L signaling pathways that lead to regulation of gene transcription. A prima ry mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involve s phosphorylation by Jak-family tyrosine kinases of one or more signal tran sducers and activators of transcription (Stat) factors which subsequently b ind 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 exc lusion of other signaling pathways. Previously, we reported that PRL direct ly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2-11 cells. In the present study, e xperiments were conducted to evaluate signaling mechanisms by which PRL reg ulates 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 FD C/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. Deoxyribon uclease I(DNase I) protection experiments revealed a motif containing a nuc lear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when ex posed to extracts from PRL-treated cells but protected by proteins from uns timulated cells. Gel mobility shift analysis of this sequence showed decrea sed protein binding after PRL stimulation. It is concluded that the PRLR in itiates pim-1 transcription by a mechanism that involves transcriptional ac tivation 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 pr esent within the promoter.