Abstract

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.

Highlights

  • 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

  • The involvement of the prostaglandin-responsive element (PGRE) and Sp1 sites in regulation by prostaglandin E1 (PGE1) was further confirmed by the increased PGE1 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

  • Effect of PGE1 and 8-Br-cAMP—To study the regulation of the human Na,K-ATPase ␤1 promoter activity by PGE1 and 8-Br-cAMP, Madin-Darby canine kidney (MDCK) cells were transiently transfected with pH␤1Ϫ1141Luc, a human Na,K-ATPase ␤1 promoter/luciferase construct [12]

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Summary

The abbreviations used are

T3, triiodothyronine; PKC, protein kinase C; PGE1, prostaglandin E1; MDCK, Madin-Darby canine kidney; PGRE, prostaglandin response element; SPGRE, Sp1-containing PGRE; 8-Br-cAMP, 8-bromo-cAMP; PMA, phorbol 12-myristate 13-acetate; CRE, cAMP-responsive element; CREM, CRE modulator; CREB, cAMP regulatory element-binding protein; CBP, CREB-binding protein; GST, glutathione S-transferase; DMEM, Dulbecco’s modified Eagle’s medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FN, fibronectin; MRE/GRE, mineralocorticoid/glucocorticoidresponsive element; TRE, thyroid hormone-responsive element; CaM kinase, Ca2ϩ/calmodulin-dependent protein kinase; ATF, activating transcription factor. The results of these studies suggest that a novel interaction between these transcription factors is involved in mediating regulation initiated by effector molecules acting through both cAMP- and Ca2ϩ-mediated pathways

EXPERIMENTAL PROCEDURES
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