Prolactin-regulated pim-1 transcription: identification of critical promoter elements and Akt signaling.

Abstract

Previously we showed that the distal element (DE) (-427 to -336 bp) within the pim-1 promoter appeared to regulate its prolactin (PRL)-induced transcription. To determine which specific DE sequences conferred PRL responsiveness, seven 12-bp deletion mutants ligated upstream of the chloramphenicol acetyltransferase gene were transfected into FDC/Nb2 cells. Results from promoter/ reporter studies showed that sequential 12-bp deletions of the DE significantly (p < 0.001) reduced PRL responsiveness. An additional site, nuclear factor-1 (-224 to -217), was also mutated by deletion or point mutation; both abrogated promoter activation by PRL (p < 0.0001). In other experiments, PRL signaling to pim-1 expression was investigated in FDC/Nb2 cells stably expressing the wild-type (WT) Jak2 cDNA or a carboxy-terminal kinase-deficient Jak2 mutant and in cells infected with adenoviral constructs of WT-Akt or dominant negative Akt. Altered Jak2 did not affect PRL-stimulated pim-1 expression while inhibition of Akt attenuated its transcription. We conclude that the DE and NF-1 half-site mediate PRL responsiveness of the pim-1 promoter. Moreover, the accumulated evidence does not support a role for the Jak2/Stat signaling pathway but, instead, implicates that Akt activation was a component of PRL-induced pim-1 transcription.