Involvement of PKA and Sp1 in the induction of p27 Kip1 by tamoxifen

Abstract

We have previously shown that tamoxifen (Tam) inhibits proliferation of estrogen receptor-negative human non-small cell lung cancer cells and this inhibition is associated with induction of p27 Kip1. In this study, we investigated the mechanism by which Tam increases p27 Kip1 expression. Because intracellular p27 Kip1 protein level is mainly controlled via posttranslational regulation, we first tested whether Tam might affect protein stability of p27 Kip1. Metabolic labeling and pulse chase assays showed that Tam did not affect the half-life of this protein. We next examined whether Tam enhanced p27 Kip1 expression through transcriptional activation. Our results demonstrated that Tam directly stimulated the p27 Kip1 promoter in lung cancer cells. Deletion and mutation analysis revealed that two Sp1 consensus sites located between −545 and −532 bp from the transcription start site were crucial for the induction of p27 Kip1 by Tam. Conversely, mutation in a CTF site (−525/−520) nearby these two Sp1 sites had little effect. Electromobility shift assays showed that Sp1 transcription factor bound to these consensus sites and the DNA binding activity of Sp1 was enhanced by Tam. Our data also demonstrated that induction of p27 Kip1 by Tam was inhibited by protein kinase A inhibitor H89, but not by protein kinase C inhibitor calphostin C and mitogen-activated kinase kinase inhibitor PD98059. Taken together, our results suggest that Tam transcriptionally activates p27 Kip1 expression via the Sp1 consensus sites in the p27 Kip1 promoter and PKA is involved in this process.