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,KATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and In this work, we present evidence that transcription of the Na,K-ATPase ␤ 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 ␤ 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 ␤ 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-␤72-167, which contains region ؊167 to ؊72 in the human ␤ 1 subunit promoter. The PGE 1 stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-␤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.

Transcription of Na,K‐ATPase alpha and beta subunit genes in renal cells is regulated by signaling initiated via G‐protein coupled receptors. Included amongst these receptors are the receptors for PGE2, the major renal prostaglandin. In the MDCK cell line PGE stimulates transcription of Na,K‐ATPase alpha and beta subunit genes. Transient transfection studies conducted in MDCK cells with a human Na,K‐ATPase β1 subunit promoter/luciferase construct, pHβ1–1141 Luc, indicate the PGE stimulation is mediated by EP1 receptors (coupled to phospholipase C) as well as EP2 and EP4 receptors (coupled to adenylate cyclase). A prostaglandin regulatory element (PGRE) within the β1 subunit promoter (−110 to −92, AGTCCCTGC) is sufficient to elicit a PGE1 stimulation, although an alternative PGRE (TGACCTTC, −445 to −438) is also a site for regulation. In addition other G‐protein coupled receptors (e.g. dopamine) as well as ionic stress mediate regulation through PGREs. Evidence for CREB binding to these PGREs has been obtained. In addition, DNA affinity precipitation, electrophoretic mobility shift, and co‐immunoprecipitation studies indicate that in addition to CBP, Sp1 is an alternative binding partner for CREB. Transducers of regulated CREB activity TORCs) are other possible alternative binding partners in mediating regulation of Na,K‐ATPase transcription through PGREs.