Gene Level Regulation of Na,K-ATPase in the Renal Proximal Tubule Is Controlled by Two Independent but Interacting Regulatory Mechanisms Involving Salt Inducible Kinase 1 and CREB-Regulated Transcriptional Coactivators

Abstract

For many years, studies concerning the regulation of Na,K-ATPase were restricted to acute regulatory mechanisms, which affected the phosphorylation of Na,K-ATPase, and thus its retention on the plasma membrane. However, in recent years, this focus has changed. Na,K-ATPase has been established as a signal transducer, which becomes part of a signaling complex as a consequence of ouabain binding. Na,K-ATPase within this signaling complex is localized in caveolae, where Na,K-ATPase has also been observed to regulate Inositol 1,4,5-Trisphosphate Receptor (IP3R)-mediated calcium release. This latter association has been implicated as playing a role in signaling by G Protein Coupled Receptors (GPCRs). Here, the consequences of signaling by renal effectors that act via such GPCRs are reviewed, including their regulatory effects on Na,K-ATPase gene expression in the renal proximal tubule (RPT). Two major types of gene regulation entail signaling by Salt Inducible Kinase 1 (SIK1). On one hand, SIK1 acts so as to block signaling via cAMP Response Element (CRE) Binding Protein (CREB) Regulated Transcriptional Coactivators (CRTCs) and on the other hand, SIK1 acts so as to stimulate signaling via the Myocyte Enhancer Factor 2 (MEF2)/nuclear factor of activated T cell (NFAT) regulated genes. Ultimate consequences of these pathways include regulatory effects which alter the rate of transcription of the Na,K-ATPase β1 subunit gene atp1b1 by CREB, as well as by MEF2/NFAT.

Keywords: CREB; CRTC; Histone Deacetylase; Na,K-ATPase; SIK1; catecholamines; kidney; proximal tubule; renal cell culture.

Figure 1

Promoter region of the human atp1b1 gene. The human atp1b1 promoter contains an MRE/GRE, an MEF regulatory element, a thyroid hormone regulatory element (TRE), an NFAT regulatory element, a TATA box, a CAAT box, three PGREs, and GC boxes which serve as binding sites for Sp1. The sequences of the PGREs are indicated, as well as a consensus CRE and a consensus Sp1 site (or GC box).

Figure 2

Structure of the human atp1a1 promoter. The human atp1a1 promoter has an MRE/GRE; AP-1, AP-2, and AP-3 sites; a CRE; and five Sp1 sites (or GC boxes).

Figure 3

CREB Structure. CREB has two glutamic acid rich domains, Q1 and Q2; a Kinase Inducible Domain (KID); and a bZIP domain, which promotes DNA binding and CREB dimerization. PKA phosphorylates CREB at Ser133 within the KID domain, which then serves as a CBP binding site. The bZIP domain mediates CREB binding to CRTCS.

Figure 4

Structure of CRTC. CRTC has an amino terminal CREB binding domain (CBD), a Regulatory Domain (REG) which can be phosphorylated at two sites (Ser171 and Ser307) by SIK1, a splicing domain (SD), and a carboxy terminal Trans Activation Domain (TAD).

Figure 5

Regulation of atp1b1 transcription by CRTCs and Class IIa HDACs. CRTCs are phosphorylated by SIK1, and translocate to the cytoplasm where they interact with 14-3-3. When SIK1 is phosphorylated and inactivated by PKA, CRTCs are then dephosphorylated by PP2B (i.e., calcineurin), provided that PP2B is activated by Ca²⁺/calmodulin. When SIK1 is phosphorylated and activated by CaMK1, SIK1 phosphorylates class IIa HDACs, which then dissociate from MEF2 transcription factors, activating transcription by MEF2. Phosphorylated class IIa HDACs translocate to the cytoplasm where they are sequestered by 14-3-3 proteins.

Figure 6

Structure of SIK1. SIK1 has an amino terminal Kinase domain that can be phosphorylated by LKB1, activating SIK1. SIK1 can also be phosphorylated at Thr322 by CaMK and activated. In contrast, SIK1 can be phosphorylated at Ser577 by PKA and inhibited.

Figure 7

Primary RPT cells have a polarized morphology with an apical surface facing the medium in vitro and a basolateral surface facing the culture dish. The cultures possess a Na⁺/Pi cotransport system (NaPi2a), a Na⁺/H⁺ antiport system (NHE3), and a Na⁺/glucose cotransport system (SGLT2) on the apical surface, facilitating Na⁺, Pi, and glucose reabsorption. The Na⁺ gradient required for reabsorption is provided by the basolateral Na⁺,K⁺-ATPase, which is also is responsible for the efflux of Na⁺ into the interstitial space. The RPT cells also have a basolateral p-AH transport system responsible for the secretion of organic anions into the lumen of the nephron. The primary RPT cells also have receptors for PTH, Ang II, dopamine, and norepinephrine.

Figure 8

Monensin treatment results in the activation of CaMK, which phosphorylates and activates SIK1. As a consequence, class IIa HDACs are phosphorylated by SIK1, and translocate to the cytoplasm, resulting in the derepression of transcription by MEF2 and NFAT transcription factors.