Regulation of Na,K-ATPase α1 and β1 subunit gene expression by low external K in neonatal rat cardiac myocytes

Abstract

Na,K-ATPase (the Na,K-pump) is an intrinsic plasma membrane enzyme that plays a critical role in many fundamental processes, such as resting membrane potentials, cell volume control and basal energy utilization. Na,K-ATPase consists of two subunits ? and ?. The ? subunit is responsible for the catalytic and transport properties, whereas the ? subunit is crucial for the correct insertion of the ? subunit in the plasma membrane. Regulation of Na,K-ATPase expression is of vital importance for basic cellular functions and reduced Na,K-ATPase function is associated with several diseases such as essential hypertension, congestive heart failure and diabetes. Decreased extracellular K is a well-established model for the study of the regulation of Na,K-ATPase expression. Low external K initially inhibits Na,K-ATPase activity due to substrate limitation; however, prolonged inhibition up-regulates ?1 and ?1 subunit gene transcription and Na,K-ATPase activity. In this study, we investigate the signal transduction pathway for up-regulation of Na,K-ATPase ?1 and ?1 subunit gene transcription by low external K using transient transfection with luciferase plasmids containing the 5’ flanking region of ?1 and ?1 subunit genes. Our results showed that the low K stimulatory effect on Na,K-ATPase ?1 and ?1 subunit gene transcription is dependent on intracellular Ca and calcineurin. The augmentation of ?1 subunit gene transcription by low K is also dependent on ERK1/2 MAPK, PKA and histone deacetylase, whereas the up-regulation of ?1 subunit gene transcription by low K is dependent on PKC, p38 and JNK MAPK. These results suggest that overlapping and distinct signal transduction pathways are involved in the low K stimulatory effect on Na,K-ATPase ?1 and ?1 subunit gene transcription. We also demonstrated that the -102 to -61 base pair region is required for the low K stimulatory effect on Na,K-ATPase ?1 subunit gene transcription. Site-directed mutation studies showed that the ATF/CRE site at -70 to -63 and the adjacent GC box at -57 to -48 in the proximal promoter region of Na,K-ATPase ?1 subunit gene are both essential for the low K response. Moreover, utilizing electrophoretic mobility shift assays, we demonstrated that transcription factors Sp1, Sp3, CREB/ATF-1 and phosphorylated CREB/phosphorylated ATF-1 bind to the low K response region and binding to the GC box and the ATF/CRE site were enhanced in response to low K. Finally, by using Western Blotting we showed that exposure of cardiac myocytes to low K increased nuclear content of Sp1, phosphorylated Sp1, Sp3 and CREB. Based on the above results, we conclude that low K-mediated up-regulation of Na,K-ATPase ?1 subunit gene transcription is dependent, in part, on ATF/CRE- and GC box-binding transcription factors.