Identification of a Prostaglandin-responsive Element in the Na,K-ATPase Beta 1 Promoter That is Regulated by CAMP and Ca2+. Evidence for an Interactive Role of CAMP Regulatory Element-binding Protein and Sp1

The Na,K-ATPase is a transmembrane protein responsible for maintaining electrochemical gradients across the plasma membrane in all mammalian cells, a process that is subject to regulation at the transcriptional as well as post-transcriptional level. Included among physiologic regulators in the kidney are prostaglandins. Previously, we demonstrated that prostaglandin E(1) (PGE(1)) increases the activity and expression of the Na,K-ATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and Allen, C. (2004) Exp. Cell Res. 299, 1-14; Taub, M. L., Wang, Y., Yang, I. S., Fiorella, P., and Lee, S. M. (1992) J. Cell. Physiol. 151, 337-346). In this work, we present evidence that transcription of the Na,K-ATPase beta(1) subunit is stimulated by PGE(1), an effect that may be mediated through the cAMP and Ca(2+) pathways. Transient transfection studies using 5'-deletion mutants of the human beta(1) subunit promoter indicated that region -100 to -92 containing the sequence AGTCCCTGC (a prostaglandin-responsive element (PGRE)) is required to elicit the stimulatory effects of PGE(1), 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and okadaic acid. Electrophoretic mobility shift assays indicated that both the cAMP regulatory element-binding protein (CREB) and Sp1 bind to the PGRE present within this region of the beta(1) subunit promoter. The involvement of the PGRE and Sp1 sites in regulation by PGE(1) was further confirmed by the increased PGE(1) stimulation that was observed following insertion of the PGRE into a promoter/luciferase construct containing a portion of a heterologous promoter and the fibronectin promoter with four GC boxes. Further evidence suggesting an interaction between Sp1 and CREB was obtained from experiments conducted with pLuc-MCS-beta 72-167, which contains region -167 to -72 in the human beta(1) subunit promoter. The PGE(1) stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-beta 72-167 was reduced when the two GC boxes immediately upstream from the PGRE were translocated farther upstream. Also consistent with an interaction between CREB and Sp1 are the results of our immunoprecipitation studies indicating that CREB co-immunoprecipitated with Sp1 when an antibody against CREB, Sp1, or the CREB-binding protein was used.