Transcriptional regulators of Na,K-ATPase subunits

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.


INTRODUCTION
Na,K-ATPase is an integral membrane protein that mainly functions as an ion pump, hydrolyzing one molecule of ATP to pump three Na + out of the cell in exchange for two K + entering the cell per pump cycle. This function is crucial to maintaining the ion gradient across the membrane and is critical for the resting membrane potential, electrical activity of muscle and nerve, Na +coupled transport, and osmotic balance and cell volume regulation (Blanco et al., 1998;Kaplan, 2002). Besides being an ion pump, Na,K-ATPase acts as a signal transducer (Xie and Askari, 2002;Pierre and Xie, 2006;Rajasekaran and Rajasekaran, 2009;Reinhard et al., 2013). Both αand β-subunit associate with various signaling molecules, including Src, phosphoinositide 3-kinase (PI3K), caveolin-1, protein phosphatase 2, and EGFR thereby activating a number of intracellular signaling pathways, including MAPK and Akt signaling, to modulate cell polarity, cell growth, cell motility and gene expression (Haas et al., 2002;Barwe et al., 2005;Cai et al., 2008;Kimura et al., 2011). Na,K-ATPase also functions as a receptor for the cardiac glycoside ouabain, a specific inhibitor of the pump. Studies with ouabain at concentrations that do not inhibit the Na,K-ATPase pump function have been particularly useful in elucidating the pump-independent signaling pathways associated with Na,K-ATPase (Xie and Askari, 2002;Silva and Soares-da-Silva, 2012;Reinhard et al., 2013). Na,K-ATPase also regulates the formation and stabilization of intercellular junctions and the β 1 and β 2 -subunits act as cell adhesion molecules (Gloor et al., 1990;Rajasekaran et al., 2001a,b;Vagin et al., 2006Vagin et al., , 2012. Na,K-ATPase is an abundantly expressed protein and can use up to about 2/3 of the energy expenditure of a cell (Howarth et al., 2012) thus requiring tight regulation. Aberrant expression and/or function of Na,K-ATPase has been associated with many disorders, with cancer being one of the more recent ones (Litan and Langhans, 2015). This review will focus on the transcriptional regulation of Na,K-ATPase by hormones, transcription factors, growth factors, extracellular stimuli, and epigenetic modification in general; followed by the role of transcription factors involved in epithelial-mesenchymal transition (EMT) and carcinoma.

THE EXPRESSION PROFILE OF Na,K-ATPase
Na,K-ATPase is a heteromeric transmembrane protein composed of an essential α-and β-subunit (Lingrel and Kuntzweiler, 1994), and an optional third subunit belonging to the FXYD proteins which are more tissue specific regulatory subunits of the enzyme (Geering, 2006). The α-subunit is the catalytic subunit responsible for transport activities of the enzyme. It contains 10 transmembrane segments; a large intracellular domain and a simple extracellular domain. An ATP-binding site and a phosphorylation site locate within the large cytoplasmic loops. The extracellular domain and the transmembrane regions harbor the binding sites for cardiac glycosides such as digoxin and ouabain (Kaplan, 2002;Sandtner et al., 2011;Laursen et al., 2013). The β-subunit is a highly glycosylated transmembrane protein, which increases the translation efficiency and stability of the α-subunit and targets the α-subunit to the plasma membrane (Blanco et al., 1998;Rajasekaran et al., 2004;Benarroch, 2011). It also modulates the affinity of Na + and K + to the enzyme (Blanco et al., 1998) and functions as a cell adhesion molecule (Gloor et al., 1990;Rajasekaran et al., 2001b;Barwe et al., 2005;Kitamura et al., 2005;Shoshani et al., 2005;Vagin et al., 2006Vagin et al., , 2012. Unlike the α-subunit, the β-subunit spans the plasma membrane only once and has a very short intracellular N-terminus and a large extracellular C-terminal domain that contains several N-glycosylation sites (Blanco et al., 1998;Benarroch, 2011). In recent years, the FXYD family has been identified as another subunit of Na,K-ATPase. They are a group of small, singlespan membrane proteins that are characterized by the FXYD motif in the extracellular domain, two conserved glycine residues within the membrane, and a serine residue at the membranecytoplasm interface (Garty and Karlish, 2006;Geering, 2008). FXYD proteins modify the affinity for Na + , K + , and ATP, pump kinetics and transport properties and stabilize Na,K-ATPase (Garty and Karlish, 2006;Geering, 2006Geering, , 2008Mishra et al., 2011).
The Na,K-ATPase subunits are highly conserved across species and among isoforms. Four isoforms of α-subunit (α 1 -α 4 ) and three isoforms of β-subunit (β 1 -β 3 ) have been identified so far, which are encoded by different genes (Blanco, 2005). In human, there is high identity in amino acid sequence among the α-subunit isoforms with 87% identity between α 2 and α 1 and 88% between α 3 and α 1 . α 4 is a little more divergent, but still sharing 79% identity with α 1 . The β-subunit isoforms are more diverse. Compared to β 1 , β 2 shows 39% identity and β 3 is 36%. Seven different FXYD proteins have been identified (FXYD1 to FXYD7), which show 43 ∼ 51% identity in their amino acid sequence.
The α-and β-subunits are expressed in a tissue specific manner (Herrera et al., 1987;Orlowski and Lingrel, 1988;Sweadner, 1989;Blanco et al., 1998;Benarroch, 2011). The α 1subunit is present in most tissues and the highest expression is found in kidney, brain and heart. Compared to the broad tissue distribution of the α 1 -subunit, the expression of other α-subunit isoforms is more restricted. The α 2 -subunit predominates in skeletal muscle, brain and heart; the α 3 -subunit is abundant in nervous tissue and heart. The α 4 -subunit was discovered as a testis-specific subunit (Shamraj and Lingrel, 1994), but now has also been found in adult human and mouse skeletal muscle (Keryanov and Gardner, 2002). Within the nervous system, the α-subunit isoforms are expressed in a cell type specific manner. For example, the α 1 -subunit is expressed in both neurons and glial cells, the α 2 -subunit is primarily found in astrocytes and oligodendrocytes and the α 3 -subunit is abundant in neurons (Benarroch, 2011). Like the α 1 -subunit, the β 1subunit is ubiquitously expressed in almost all tissues with the greatest levels in brain and kidney (Orlowski and Lingrel, 1988). The β 2 -subunit is mainly found in nervous tissue, kidney and heart (Avila et al., 1998), whereas the β 3 -subunit predominates in kidney and adrenal gland but is also presents in brain, lung and liver (Malik et al., 1998). The FXYD proteins show a tissue-specific distribution as well. FXYD1 (phospholemman) is expressed in heart and skeletal muscle; FXYD2 (γ-subunit) in kidney; FXYD3 (MAT-8) in stomach and colon; FXYD4 (CHIF) in kidney and colon; FXYD5 (dysadherin) in intestine, lung and kidney; FXYD6 and FXYD7 in brain (Garty and Karlish, 2006;Geering, 2006). In addition, the expression levels of α-and βsubunit change during development. For example, in brain, all three α-subunit isoforms increase in abundance from fetus to adult (Orlowski and Lingrel, 1988). However, the α 1 -subunit is more abundant in fetal kidney and heart than in adult tissues (Herrera et al., 1987). In rat heart, the α 3 -subunit decreases sharply after birth and is replaced by α 2 -subunit in adults (Zahler et al., 1996). The β 1 -subunit increases in the early stage of embryonic development, but decreases to the basal level during embryo implantation (Deng et al., 2013). With these diverse tissue distributions of Na,K-ATPase subunit isoforms, multiple Na,K-ATPase isozymes can form, rendering cells able to respond more precisely to extracellular stimuli. For instance, while in kidney α 1 β 1 is the major αβ complex, all combinations of αβ complexes have been found in heart and brain (Mobasheri et al., 2000).
The spatial and temporal control of Na,K-ATPase expression occurs at the transcriptional, post-transcriptional, translational and post-translational level. Here, we will review findings on the transcriptional regulation of Na,K-ATPase over the past decades ( Table 1). Extracellular stimuli, such as growth factors play a key role in the regulation of Na,K-ATPase by activating intracellular signaling pathways including protein kinase A (PKA), protein kinase C (PKC), and other Ca 2+ -regulated signaling pathways. Numerous DNA-binding-site-specific transcription factors have been reported to be involved in the regulation of Na,K-ATPase expression, including hormone receptors, Snail1, specificity protein (Sp) and Zinc finger E-box-binding homeobox 1 (ZEB1). Recently, it has been shown that hypermethylation in promoter regions of human ATP1B1 gene is associated with reduced expression of β 1 -subunit (Selvakumar et al., 2014), indicating that epigenetic modification might be another mechanism of transcriptional regulation of Na,K-ATPase subunits.
PROMOTER ANALYSIS OF Na,K-ATPase α-AND β-SUBUNITS Transcription factors activate or repress Na,K-ATPase expression by binding to specific DNA elements on the promoter regions. There are numerous sequence-specific elements on the promoter regions of the various Na,K-ATPase subunits, of which β 1 has been extensively studied (Figure 1).
The α 1 -subunit is encoded by the gene ATP1A1. The ATP1A1 promoter has very high GC content, which is a common characteristic of all housekeeping genes. It contains a potential TATA box (−27 bp; Shull et al., 1990), two mineralocorticoid/glucocorticoid response element (MRE/GRE) sites (−598 to −574 bp and −252 bp; Kolla et al., 1999); potential binding sites for AP-1(−507 and −453 bp), AP-2 (−568 and −380 bp), AP-3 (−286 bp), and several CTF/NF-1 and potential Sp1 binding sites (Shull et al., 1990). The nucleotide sequence from around −100 bp to the transcription initiation site is highly conserved among the human and rat Atp1a1 genes (Kobayashi et al., 1997). An asymmetrical ATF/CRE site (−77 to −64 bp) is found in the rat Atp1a1 promoter (Kobayashi and Kawakami, 1995). In addition, the rat Atp1a1 promoter contains a Sp1 consensus sequence (−56 to −51 bp) and a Sp1 binding site (−120 to −106 bp; Suzuki- . The α 2 -subunit is encoded by the gene ATP1A2. The promoter region of human ATP1A2 contains a potential TATA box, two potential Sp1 recognition sites, numerous potential AP-1, AP-2, and NF-1 binding sites, and several sequences that are similar to the glucocorticoid receptor binding site (Shull et al., 1989;Malyshev et al., 1991). The major positive regulatory elements are located at the position from −175 to −108 bp on the rat Atp1a2 gene, harboring two E boxes (−144 to −139 bp and −135 to −130 bp), a Spl binding consensus sequence (−123 to −118 bp) and a Sp1 binding site (−114 to −109 bp; Ikeda et al., 1993).
The α 3 -subunit is encoded by the gene ATP1A3. The human ATP1A3 promoter has high GC content, but no conventional TATA box (Malyshev et al., 1991). Sequence analysis reveals several potential Sp-1 binding sites and AP-1 sites, two AP-2 sites, two AP-4 sites, three GRE elements, two CACCC sequences, a NF-1 binding site, a couple of TRE elements (Pathak et al., 1990), and the binding sites for CREB/ATF and NF-Y/C-EBP (Benfante et al., 2005). These sites are well conserved between the human and rat Atp1a3 gene (Pathak et al., 1990). It was subsequently shown that two Sp1 binding sites (−110 to −100 bp; −59 to −47 bp), a CCAAT box (−64 to −61 bp) and a half CRElike site (−87 to −83 bp) are functional. Mutations in the Sp1 binding site (−110 to −100 bp) or the CCAAT box significantly reduced the ATP1A3 promoter activity and these two sites worked synergistically in inducing ATP1A3 promoter activity. The CRE-like element itself did not affect ATP1A3 promoter activity, but cooperated with the upstream Sp1 site (Benfante et al., 2005). Three TRE elements (−636 to −457 bp; −218 to −106 bp; and −106 to −6 bp) showed strong and specific interaction with thyroid hormone receptor (TR) and modulated ATP1A3 promoter activity (Bajpai et al., 2001). In addition, the region from −210 bp to the transcription initiation site was sufficient to direct brain-specific expression of the rat α 3 -subunit (Pathak et al., 1994).
The α 4 -subunit is encoded by the gene ATP1A4. In silicon sequence analysis of putative promoter regions of human and mouse ATP1A4 showed that they do not contain a TATAbox sequence, but have potential binding sites for transcription factors AP-1, AP-4, GATA3, NF-Y, MYOD, NF-1, NFAT (Keryanov and Gardner, 2002) and two consensus CRE sites (Rodova et al., 2006). However, ATP1A4 does not have any Sp1 binding site that is common on the promoter regions of other subunits (Keryanov and Gardner, 2002).  Mony et al., 2013), three thyroid hormone response elements (TRE; Feng et al., 1993), three progesterone element (PRE) half sites (Cochrane et al., 2012) and several Sp1 binding sites (Matlhagela et al., 2005;

Regulator
The roles in regulating Na, K-ATPase transcription References
The β 2 -subunit is encoded by the gene ATP1B2. The region from −280 bp to the transcriptional initiation site on the promoter of ATP1B2 gene is highly conserved between rat, mouse and human. The human ATP1B2 promoter contains a consensus TATA sequence at position −94 bp and a Sp1 binding site at position −120 bp (Avila et al., 1998). The mouse Atp1b2 promoter has a TATA-like sequence (−23 to −28 bp), a putative CAAT box (−137 to −142 bp), and a GC box (−144 to −149 bp; Shyjan et al., 1991). Sequence analysis revealed a potential TATA box (−29 bp); two Sp1 binding sites (−55 and −147 bp) and a serum response element (SRE: −263 bp) on the rat Atp1b2 promoter region . In addition, an AMOG regulatory element (AMRE: GAGGCGGGG at position −87 to −79 bp) and a functional Sp1 binding site (−147 to −142 bp) were found in rat Atp1b2 promoter regions .
The β 3 -subunit is encoded by the gene ATP1B3. The human ATP1B3 promoter contains a TATA-like sequence (−413 to −408 bp) and two Sp1-like elements (−323 to −315 bp and −312 to −304 bp). Moreover, the human ATP1B3 also has four identical 32-nucleotide-long repeats from position −760 to −633 bp. The sequence from −1 to −328 bp on the 5 ′ flanking region of human and mouse Atp1b3 shows high homology. The mouse Atp1b3 promoter has one Sp1-like sequence (−319 to −310 bp), but does not have any TATA-like sequence. No CAAT or GC boxes are present at the promoter region of human or mouse Atp1b3 (Malik et al., 1998).

TRANSCRIPTIONAL REGULATION OF Na,K-ATPase BY HORMONES AND NUCLEAR RECEPTORS
Hormones are major regulators of Na,K-ATPase expression.

Mineralocorticoid Receptor (MR) and Glucocorticoid Receptor (GR)
MR and GR are members of the nuclear receptor family. They have three major functional domains: a N-terminal regulatory domain (NTD); a DNA-binding domain (DBD) and a ligand-binding domain (LBD; Kumar and Thompson, 2005). The MR is highly homologous to the GR in the DBD and LBD but not in the NTD (Kolla et al., 1999) and while mineralocorticoids only activate the MR, glucocorticoids activate both GR and MR. The activated MR and GR translocate into the nucleus and bind to a specific palindromic DNA sequence known as glucocorticoid/mineralocorticoid response element (GRE/MRE) located in the promoter regions of target genes, thus modulating gene transcription. The GRE is composed of imperfect inverted hexanucleotide repeats separated by three nucleotides (GGTACA NNN TGTTCT; Nordeen et al., 1990). Glucocorticoids regulate the transcription of Na,K-ATPase in a variety of tissues including lung, kidney, liver, heart, spinal cord, cardiac muscle and smooth muscle (Orlowski and Lingrel, 1990;Bhutada et al., 1991;Celsi et al., 1991;Wang and Celsi, 1993;González et al., 1994González et al., , 1996Wang et al., 1994;Muto et al., 1996;Barquin et al., 1997;Ingbar et al., 1997;Chalaka et al., 1999;Dagenais et al., 2001;Hao et al., 2003b). This regulation is specific to the cell type and the developmental stage. For example, dexamethasone, a synthetic potent glucocorticoid, increased the mRNA levels of α 1 -and β 1 -subunit in a rat liver cell line and in a fetal rat lung epithelial cell line (Bhutada et al., 1991;Chalaka et al., 1999). However, in cultured neonatal rat cardiac myocytes, dexamethasone only induced α 2 -subunit mRNA, and α 1 -, α 3 -, and β-subunit transcripts remained unchanged (Orlowski and Lingrel, 1990). Injection of betamethasone, another synthetic analog of glucocorticoid, increased α 1 -and β-subunit mRNA abundance in 10-day-old rats, but not in adult rats (Celsi et al., 1991).
The promoter region of the ATP1B1 gene contains three potential MRE/GREs at nucleotide position −1048 to −1027 bp; −650 to −630 bp and −276 to −271 bp. The first two contain two half-binding sites separated by 10 and 9 base pairs, respectively, whereas the last one consists of a single half-site. Although all three MRE/GREs were involved in the induction of ATP1B1 promoter activity by corticoids, the one at position −650 to −630 bp was the strongest (Derfoul et al., 1998). Both MR and GR bound to the GRE/MRE (−650 to −630 bp) and activated the transcription from a heterologous neutral promoter in response to corticoids (Derfoul et al., 1998;Hao et al., 2003b). A similar element was found in the human ATP1A1 promoter region at position −598 to −574 bp in which two half sites are separated by 12 base pairs. This MRE/GRE bound both MR and GR and activated a heterologous neutral promoter activity (Kolla et al., 1999). In addition, a GRE (+434 to +448 bp) in the coding region of the rat Atp1b1 gene has been reported in a rat lung epithelial cell line. This element acted as an enhancer and increased the transcription from a heterologous promoter by dexamethasone (Hao et al., 2003a).

Progesterone Receptor
Progesterone receptor (PR) also belongs to the nuclear receptor family. PR's sequence and architecture is very similar to MR and GR (Leo et al., 2004). Like MR and GR, PR is activated by its ligand progesterone and then translocates into the nucleus, forms a homodimer and then binds to specific progesterone response elements (PREs) within the promoter of target genes to modulate transcription (Leonhardt et al., 2003). Progesterone increased the mRNA and protein levels of β 1 -subunit (Cochrane et al., 2012;Deng et al., 2013), and this up-regulation was enhanced by estradiol-17β (Deng et al., 2013). RU486, a PR antagonist, completely blocked the increase in β 1 -subunit mRNA by progesterone (Deng et al., 2013). In addition, human β 1subunit was up-regulated by both PR-A and PR-B, two different PR isoforms (Richer et al., 2002). Moreover, PR bound to three PRE half sites on the ATP1B1 promoter; Progestin, a synthetic progestogen with similar effect to progesterone, induced ATP1B1 promoter activity, an induction that was suppressed by RU486 (Cochrane et al., 2012).

Insulin
Insulin is an important regulator of Na,K-ATPase. It binds to insulin receptor and activates various cellular signaling pathways, thus regulating cell functions. Insulin modulates Na,K-ATPase activity by increasing Na + influx; increasing the affinity of Na,K-ATPase for intracellular Na + ; altering phosphorylation status of Na,K-ATPase and facilitating the translocation of intracellular Na,K-ATPase to the plasma membrane. In addition, insulin also regulates the transcription, translation and stability of Na,K-ATPase (Ewart and Klip, 1995;Lavoie et al., 1996;Sweeney and Klip, 1998;Therien and Blostein, 2000;Clausen, 2003). The transcriptional regulation of Na,K-ATPase by insulin occurs in an isoform specific manner. For example, insulin selectively increased α 2 , but not α 1 mRNA level in vascular smooth muscle cells (VSMC) and 3T3-L1 cells (Russo and Sweadner, 1993;Tirupattur et al., 1993;Sweeney and Klip, 1998). However, it reduced the β 1 mRNA level (Russo and Sweadner, 1993).

Specificity Protein
Specificity protein (Sp) is a small family of zinc-finger transcription factors with four members: Sp1, Sp2, Sp3, and Sp4. Sp1, Sp2, and Sp3 are ubiquitously expressed, while Sp4 is expressed mainly in neurons and testes. The Sp family members contain three zinc fingers close to the Cterminus, which is DBD, and preferentially bind to the GC box [(G/T)(G/A)GGC(G/T)(G/A)(G/A)(G/T)] on the promoter regions of target genes to enhance gene expression. Sp1, Sp3, and Sp4 have a highly conserved DBDs and they recognize the same consensus GC rich element with identical affinities. However, Sp2, in which the important histidine residue within the first zinc finger is replaced by a leucine residue, does not bind to the GC box but instead binds to a GT rich element (Suske, 1999). Potential Sp1 binding sites were found on the promoter regions of almost all Na,K-ATPase subunits, including Atp1a1, Atp1a2, Atp1a3, Atp1b1, and Atp1b2 (Shull et al., 1989(Shull et al., , 1990Kawakami et al., 1990;Yagawa et al., 1990;Malyshev et al., 1991;Suzuki-Yagawa et al., 1992;Ikeda et al., 1993;Yu et al., 1996;Avila et al., 1998;Benfante et al., 2005). It has been shown that Sp1 and Sp3 bind to GC boxes on the promoter regions of rat Atp1a1 (Kobayashi et al., 1997) and Atp1b1 (Murakami et al., 1997). Mutations in the GC box sequences decreased basal Atp1b1 transcription in neonatal rat cardiac myocytes . Sp1, Sp3 and Sp4 also bind to two Sp1 binding sites (−110 to −100 bp; −59 to −47 bp) on human ATP1A3 promoter. Mutations in Sp1 binding site (−110 to −100 bp) significantly reduced the promoter activity (Benfante et al., 2005). Recently, Sp1, Sp3, and Sp4 had been shown to increase α 1 -, α 3 -, and β 1 -subunit transcription in murine neurons by binding to their promoter regions (Johar et al., 2012). Mutating the Sp binding sites on Atp1a1, Atp1a3, and Atp1b1 promoters led to a significant decrease in their promoter activity. Silencing Sp1, Sp3, or Sp4 significantly decreased the mRNA levels of α 1 -, α 3 -, and β 1 -subunit, while overexpressing Sp1, Sp3, and Sp4 increased their transcription (Johar et al., 2012). Sp1 also binds to two Sp binding sites located near its TATA box on rat Atp1b2 promoter and enhanced the expression of β 2 -subunit. In addition, the functional Sp-binding site on Atp1a1 promoter is well conserved in rat brain, kidney and liver Kobayashi et al., 1997;Nemoto et al., 1997), indicating that it might contribute to the constitutional expression of α 1 -subunit. The Sp binding sites are also required for the regulation of Na,K-ATPase transcription in response to extracellular stimuli. For example, hyperoxia selectively upregulated β 1 -subunit transcription in MDCK cells (Wendt et al., 1998). Hyperoxia increased the binding of Sp1 and Sp3 to the Atp1b1 promoter and deletion of a GC rich element resulted in loss of both basal and hyperoxia-activated transcription . Sp might also augment transcriptional activation of Na,K-ATPase through other transcription factors. For example, Sp1 significantly enhanced MR and GR expression (Kolla et al., 1999) and elevated MR and GR might synergize with Sp1 to upregulate Na,K-ATPase transcription.

Snail
Snail1 is the first identified member of the Snail family, which belongs to the zinc-finger transcription factors and has three members in vertebrates: Snail1, Snail2, and Snail3. The Snail family has a highly conserved C-terminal region, which is the DBD containing four to six zinc fingers; and a divergent Nterminal region, which contains a SNAG (Snail/Gfi) domain and recruits the transcription repressor complex (such as sin3A/HDAC). The Snail family functions as a transcriptional repressor by binding to the E-box (CAGGTG) on the promoter regions of target genes which are important for embryonic development as well as tumor progression (Nieto, 2002). Four Eboxes and a non-canonical E-box are present on the promoter region of the ATP1B1 gene. Snail1 binds to the non-canonical E-box located at position −71 to −66 bp and selectively repressed the expression of β 1 -subunit without affecting the α 1 -subunit level . Overexpression of Snail1 repressed ATP1B1 promoter activity in a dose dependent manner and markedly reduced the level of β 1 -subunit in MCF7 and MDCK cells. Knock-down of Snail by RNA interference increased the abundance of β 1 -subunit mRNA .

Activating Transcription Factor 1 (ATF-1)/cAMP Response Element Binding Protein (CREB) Family
ATF/CREB belongs to the basic leucine zipper factor (bZIP) family, which bind to DNA as dimers and function as activators. The ATF/CREB family is composed of different ATFs, CREB, CREM (cAMP response element modulator) and related proteins. This family contains an N-terminal DBD and a C-terminal bZIP domain that binds other bZIP transcription factors to form homo-and heterodimers. Both ATF and CREB bind to the cAMP response element (CRE: GTGACGT A/C A/G). Phosphorylation of ATF-1 and CREB by multiple kinases enhances the binding to CRE and activates the transcription of target genes which play an important role in cell survival and cell growth (Hai and Hartman, 2001). Dibutyryl cAMP (db-cAMP), a cell-permeable cAMP analog that activates cAMP dependent protein kinase (PKA) and phosphorylates the CREB family, induced the mRNA of α 1 -subunit but not β 1 -subunit (Dagenais et al., 2001). The rat Atp1a1 promoter has an asymmetrical ATF/CRE site at position −70 to −66 bp. The ATF-1/CREB heterodimer bound to this site and induced constitutive expression of α l -subunit. Blocking the binding of ATF-1/CREB to the CRE site reduced α 1 -subunit transcription (Kobayashi and Kawakami, 1995). Phosphorylated ATF-1 increased rat α 1subunit transcription and dephosphorylation of the ATF-1/CREB heterodimer by alkaline phosphatase reduced its binding to the CRE site on rat Atp1a1 gene (Kobayashi et al., 1997). In addition, human ATP1A4 promoter region has consensus CRE sites. db-cAMP and ectopic expression of CREMtau, a testis specific splice variant of CREM activated the ATP1A4 promoter activity. This activation required the CRE site located at −263 bp relative to the transcription initiation site (Rodova et al., 2006).

Zinc Finger E-Box Binding Homeobox 1 (ZEB1)
The ZEB family has two members in vertebrates: ZEB1 (also known as Areb6) and ZEB2. They contain five major domains: two zinc-finger clusters separated by a central homeodomain, a Smad interacting domain (SID) and a CtBP interacting domain (CID). Each zinc finger cluster contains 3 or 4 zinc fingers and is responsible for DNA binding. ZEB binds to E-boxes (CACCT and CACCTG) on the promoter of target genes and recruits coactivators (PCAF or p300), thus activating gene transcription (Vandewalle et al., 2009). The rat Atp1a1 promoter has a consensus Areb6 (ZEB1) binding sequence and Areb6 (ZEB1) had been shown to activate gene transcription in a cell-specific manner . High-fat diet also induced the binding of ZEB to rat Atp1α1 promoter and increased the mRNA and protein level of α 1 -subunit in nuclear extracts from gastrocnemius muscle (Galuska et al., 2009). Moreover, Cpeptide increased the binding of ZEB to rat Atp1α1 promoter region. Partial silencing of ZEB markedly reduced the effect of Cpeptide on the increase in α 1 -subunit expression (Galuska et al., 2011). In addition, ZEB binding sites were also found on FXYD1 promoter at positions −270 to −295 bp and −329 to −354 bp (Galuska et al., 2009).

CCAAT Box Binding Transcription Factors (CBFs)
CCAAT box is one of the most common elements in eukaryotic promoters, which is often found between -60 to -100 bp relative to the transcription initiation site. Several transcription factors have been reported to bind to the CCAAT box, including NF-Y, NF-1, and CCAAT/enhancer-binding protein (C/EBP) isoforms (CEBP-α, CEBP-β, CEBP-γ, CEBP-δ, and CEBP-ε), which are essential for optimal transcriptional activation of target genes (Mantovani, 1998). CCAAT boxes have been found in Atp1a2, Atp1a3, and Atp1b1 promoter regions (Pathak et al., 1990;Malyshev et al., 1991;Benfante et al., 2005;Deng et al., 2013). The CCAAT box (−61 to −64 bp) on human ATP1A3 promoter is well conserved between rat and human and NF-YB had been shown to bind to this CCAAT box. The presence of the CCAAT box significantly increased ATP1A3 promoter activity in both neuron and non-neuron cell lines, while mutations in the CCAAT box reduced the promoter activity (Benfante et al., 2005). In addition, the CCAAT/enhancer binding protein beta (Cebpb) might be involved in regulating β 1 -subunit expression in human luminal epithelial cells. The active isoform of Cebpb significantly increased Atp1b1 promoter activity, but the truncated isoform of Cebpb had no effects on Atp1b1 promoter activity (Deng et al., 2013).

GROWTH FACTORS AND TRANSCRIPTIONAL REGULATION OF Na,K-ATPase
Transforming Growth Factor-β Transforming growth factor-β (TGF-β) is a secreted protein that regulates numerous physiological processes, including cell differentiation, proliferation, development and survival. TGF-β has three isoforms in mammals: TGF-β 1 , TGF-β 2 , and TGFβ 3 , which are encoded by different genes. Aberrant TGF-β signaling has been linked to cancer initiation, invasion and metastasis (Lamouille et al., 2014). TGF-β treatment reduced mRNA and protein levels of Na,K-ATPase α-and β-subunits in primary cultures of renal proximal tubule cells (Tang et al., 1995). However, TGF-β 1 seems to regulate Na,K-ATPase subunit expression in a cell type specific manner. Some studies showed that TGF-β 1 reduced β 1 -subunit as well as α 1 -, α 2 -, and α 3subunit mRNA levels in young FRTL-5 rat thyroid cells (Pekary et al., 1997). Studies from our group found that although TGFβ 1 decreased the surface levels of β 1 -subunit in kidney cells, this seemed to occur at the post-translational level since no significant changes in mRNA levels were observed between control and TGF-β 1 -treated cells (Rajasekaran et al., 2010). TGFβ 1 also prevented the increase in α 1 -subunit mRNA induced by steroid hormones (Husted et al., 2000). Nevertheless, TGF-β 2 selectively decreased the transcription of β 1 -subunit in ARPE-19 cells, a human retinal pigmented epithelial cell line, involving two transcription factors: hypoxia inducible factor (HIF) and Smad3 (Mony et al., 2013). HIF belongs to the basic helix-loop-helix (bHLH) superfamily and is a heterodimer composed of a hypoxia induced HIF-α subunit and a constitutively expressed HIF-1β subunit. Three HIF-α subunits have been identified so far: HIF-1α, HIF-2α, and HIF-3α. HIF-1α contains three major domains: a N-terminal domain, which is a DBD containing a bHLH; an oxygen-dependent degradation domain (ODDD) that mediates oxygen-regulated protein stability and a C-terminal domain, which recruits transcriptional coactivators such as CBP/p300 to activate gene transcription. The HIF complex associates with hypoxia response elements (HREs) in the regulatory regions of target genes and binds the transcriptional coactivators to induce gene expression. Besides hypoxia, HIF-1 is also regulated in an oxygen-independent manner (Ke and Costa, 2006). TGF-β 2 increased HIF-1α expression in ARPE-19 cells and blocking the binding of HIF-1α to HREs with echinomycin prevented the TGF-β 2 -induced decrease of the β 1 -subunit (Mony et al., 2013). Since HIF-1α generally functions as a transcriptional activator, this observation raised the possibility that other transcription repressors may antagonize the function of HIF-1α in the regulation of Na,K-ATPase expression. The ATP1B1 promoter region also contains a SBD in close proximity to a putative HRE. Smads are transcription modulators usually activated by TGF-β signaling that can inhibit gene expression by preventing the action of transcriptional activators (Massague et al., 2005). Studies in ARPE19 cells indeed support the idea that both Smad3 and HIF-1α cooperated in regulating the expression of β 1 -subunit expression by TGF-β 2 (Mony et al., 2013).

Fibroblast Growth Factor
Fibroblast growth factors (FGFs) control a wide range of biological functions and regulate cellular proliferation, survival, migration, and differentiation. FGFs are a family of glyocoproteins that are generally sequestered by the extracellular matrix and cell surface heparan sulfate proteoglycans. Activation of FGF signaling occurs upon release of the factor and binding to one of the four highly conserved FGF receptors, FGR1, FGR2, FGR3, and FGR4 (Turner and Grose, 2010). Incubation of cells in serum rapidly increased α 1 -and β 1 -subunit mRNA levels in VSMC and rat liver cells and induced Atp1a1 and Atp1b1 promoter activity, suggesting that serum regulates Na,K-ATPase gene expression at the transcriptional level (Kirtane et al., 1994;Nemoto et al., 1997). Serum differentially regulated α 1 -and β 1 -subunit mRNA induction and activation of PKC and tyrosine kinase activity was required for up-regulation of α 1 -subunit mRNA but not for β 1 -subunit. Further studies revealed that FGF treatment stimulated Atp1a1 and Atp1b1 promoter activities, similar to the observation in serum treated VSMC (Nemoto et al., 1997). Furthermore, keratinocyte growth factor (KGF), a member of the FGF family and a polypeptide mitogen secreted by fibroblasts and endothelial cells that acts primarily on epithelial cells, increased the abundance of α 1 -subunit, but not β 1 -subunit mRNA in alveolar type II cells .

PROSTAGLANDIN AND Na,K-ATPase TRANSCRIPTION
Prostaglandin E1 (PGE1) is a member of the prostaglandin family, which are lipid mediators produced from arachidonic acid by cyclooxygenase and prostaglandin synthases. Prostaglandins exert their effects by activating G protein-coupled receptors and four PGE receptors have been reported: EP1 (E prostanoid receptor 1), EP2, EP3, and EP4 (Sugimoto and Narumiya, 2007). Prostaglandins are important regulators of ion transport in the kidney (Bonvalet et al., 1987) and PGE1 increased the transcription of α-and β-subunit of Na,K-ATPase in MDCK cells (Taub et al., 1992(Taub et al., , 2004Matlhagela and Taub, 2006). The increase in the transcription of β 1 -subunit induced by PGE1 was mediated by EP1 and EP2 receptors and PGE receptor antagonists inhibited the promoter activity of human ATP1B1 induced by PGE1 (Matlhagela and Taub, 2006). Three prostaglandin response elements (PGRE) have been identified within the human ATP1B1 promoter, which are located at position −445 to −438 bp (PGRE1: TGACCTTC); −226 to −216 bp (PGRE2: GTCCCTCA); and −92 to −100 bp (PGRE3: AGTCCCTGC; Matlhagela et al., 2005;Matlhagela and Taub, 2006) and PGRE1 is well conserved among species (Matlhagela and Taub, 2006). The PGRE sequence is similar to the consensus CRE, and CREB had been shown to bind to these sequences indicating that PGREs might be cAMP response elements. The binding affinity of CREB to three PGREs was varied with PGRE3 > PGRE1 > PGRE2 (Matlhagela et al., 2005). Sp1, Sp3 as well as CREB all bound to PGRE (Matlhagela and Taub, 2006) and the increase in transcription of β 1 -subunit was mediated through CREB binding to PGRE1 and PGRE3 as well as Sp1 binding to an adjacent Sp1 site (Matlhagela et al., 2005;Matlhagela and Taub, 2006). Mutations in PGRE3 or the GC box (−118 to −112 bp) immediately downstream of PGRE3 failed to mediate the increase in ATP1B1 promoter activity induced by PGE1 (Matlhagela and Taub, 2006). Besides PGE1, PGE2 and PGF2α also have been shown to induce ATP1B1 promoter activity in primary renal proximal tubule (RPT) cells (Herman et al., 2010).

Na,K-ATPase ACTIVITY AND TRANSCRIPTIONAL REGULATION
Chronic inhibition of Na,K-ATPase activity by ouabain leads to an increase in the abundance of Na,K-ATPase. For example, ouabain increased the mRNA levels of α 1 -and β 1 -subunit in cultured rat astrocytes (Hosoi et al., 1997;Muto et al., 2000), which was abolished by actinomycin D, a transcription inhibitor acting by interfering with mRNA synthesis (Muto et al., 2000). Ouabain also regulated the transcription of α 3 -and β 1 -subunit in cultured neonatal rat cardiac myocytes. Ouabain augmented the Atp1b1 promoter activity and the effect of ouabain on the regulation of Na,K-ATPase subunits was dependent on extracellular Ca 2+ and calmodulin (Kometiani et al., 2000). The inhibition of Na,K-ATPase activity by ouabain can be mimicked by incubating cells in a low K + environment and low extracellular K + increased α 1 -and β 1 -subunit mRNA levels in rat cardiac myocytes (Qin et al., 1994;Zhuang et al., 2000;Wang et al., 2007a). Sp and CREB family transcription factors were required for the up-regulation of Na,K-ATPase subunits induced by low K + . Rat Atp1a1 promoter region has a CRE/ATF site (at −70 to −63 bp) and a GC box motif (at −57 to −48 bp). Mutations in the CRE/ATF site or GC box substantially reduced low K + -mediated Atp1a1 promoter activity. Low K + increased the expression of Sp1, Sp3 and CREB-1 and enhanced the binding of these transcription factors to the GC box and, to a lesser extent, to the CRE/ATF site on rat Atp1a1 promoter (Wang et al., 2007b). The sequences between -102 to +151 bp on rat Atp1b1 were required for low K + -induced trans-activation of reporter gene expression (Qin et al., 1994). Low K + enhanced the binding of Sp1 and Sp3 to GC box elements on the Atp1b1 promoter . Mutations in potential GC box sequences decreased basal and low K + -mediated up-regulation of β 1 -subunit transcription in neonatal rat cardiac myocytes . Inhibiting the binding of Sp1 and Sp3 to GC boxes by mithramycin, which has high affinity for GC-rich DNA sequences (Matlhagela et al., 2005), blocked low K + -mediated up-regulation of Atp1a1 and Atp1b1 promoter activity. Multiple cellular signaling pathways were involved in this low K + -induced increase in the transcription of α 1 -and β 1 -subunits. Protein kinase A (PKA), ERK1/2, and histone deacetylase (HDAC) were required for up-regulation of α 1 -subunit transcription, whereas the transcription of β 1 -subunit was dependent on protein kinase C (PKC), c-Jun-N-terminal kinase (JNK) and p38 mitogenactivated protein kinase (MAPK; Wang et al., 2007a). However, it is important to point out that inhibition of Na,K-ATPase and therefore an increase in intracellular sodium regulates Na,K-ATPase on multiple levels including transcription, transport, endocytosis and degradation (Vinciguerra et al., 2003(Vinciguerra et al., , 2004Wang et al., 2014).
NEURONAL ACTIVITY AND Na,K-ATPase TRANSCRIPTIONAL REGULATION α 1 -, α 3 -, and β 1 -subunits are abundantly expressed in most neurons in the central nervous system and neuronal activity modulates the transcription of Na,K-ATPase subunits. Depolarization by KCl increased the promoter activities as well as the mRNA levels of α 1 -, α 3 -and β 1 -subunits; however, impulse blockade induced by tetrodotoxin (TTX), a voltagedependent Na + channel blocker, significantly reduced α 1 -, α 3 -, and β 1 -subunit transcripts in murine neurons (Johar et al., 2012(Johar et al., , 2014. At least two transcription factors were involved in this activity-dependent transcriptional regulation in neurons, nuclear respiratory factor 1 (NRF1) and the Sp family (Johar et al., 2012(Johar et al., , 2014. NRF1 is a transcription factor which was first identified as an activator of cytochrome C (Evans and Scarpulla, 1989). NRF1 increases the expression of nuclear genes required for mitochondrial biogenesis and function, but also binds to the promoter region of target genes which control cell cycle, cell growth, cell adhesion, migration, and tumor invasion (Okoh et al., 2011). Increased NRF1 levels have been observed in hepatoma, thyroid oncocytoma and breast cancers (Dong et al., 2002;Savagner et al., 2003;Okoh et al., 2011). The consensus NRF1 binding site is (T/C)GCGCA(C/T)GCGC(A/G), which is a GC-rich element. In addition, NRF1 binds to sequences with an invariant GCA core flanked by GC-rich regions, which can be found in 5 ′ flanking regions of Atp1a1, Atp1a3, and Atp1b1 genes. It has been reported that NRF1 binds to the promoter regions of Atp1a1 and Atp1b1 in murine neurons at binding sites that are conserved among mice, rats and humans. Mutations in the NRF1 binding sites significantly decreased Atp1b1 promoter activity, but increased Atp1a1 promoter activity, which indicates that NRF1 differentially regulates Na,K-ATPase subunits. Indeed, silencing of NRF1 significantly decreased β 1 -subunit transcript but increased the mRNA level of α 1 -subunit and overexpression of NRF1 increased the mRNA level of the β 1 -subunit but decreased the α 1 -subunit transcript. NRF1 itself is regulated by neuronal activity (Yang et al., 2006). Depolarization by KCl increased NRF1 expression, and TTX reduced the NRF1 expression (Johar et al., 2012) and knockdown of NRF1 with small interference RNA blocked the up-regulation of the β 1 -subunit and down-regulation of the α 1 -subunit induced by KCl, whereas overexpression of NRF1 rescued the down-regulation of the β 1 -subunit and upregulation of the α 1 -subunit by TTX. The inverse regulation of Na,K-ATPase α 1 -and β 1 -subunit by NRF1 in response to KCl and TTX points to additional transcription factors and an intricate network regulating Na,K-ATPase expression by neuronal activity.

EPIGENETIC MECHANISMS IN THE REGULATION OF Na,K-ATPase TRANSCRIPTION
DNA methylation is an important epigenetic mechanism in the regulation of gene expression. Methylation generally occurs at the cytosine bases of CpG sequences throughout the entire genome, with the exception of CpG islands usually found in gene promoter regions (Cedar and Bergman, 2009). Methylation patterns often depend on tissue type and developmental stage, correlating with transcriptional activity. Aberrant DNA methylation patterns have been linked to altered gene expression in various genetic diseases and tumors (Bird, 2002). Higher methylated CpGs were found in the first exon of Atp1a3, which correlated with the lower expression of this gene in pig liver (Henriksen et al., 2013). Manganese exposure led to sustained Atp1a3 promoter hypermethylation and downregulation of its transcript level in mice . CpG islands are also found in ATP1B1 and Atp1b2 promoter regions (Alvarez de la Rosa et al., 2002;Selvakumar et al., 2014), which are conserved between human and mice and remain unmethylated (Alvarez de la Rosa et al., 2002). However, ATP1B1 promoter is hypermethylated in tumor samples of clear cell renal cell carcinoma, which showed reduced β 1 -subunit levels (Selvakumar et al., 2014). Knock-down of the tumor suppressor gene von Hippel-Lindau (VHL) enhanced ATP1B1 promoter methylation and decreased β 1 -subunit expression, while inhibition of methyltransferase by 5-Aza-2 ′ -deoxycytidine rescued β 1 -subunit mRNA expression in VHL-knockdown cells (Selvakumar et al., 2014).
Methylation is also involved in transcriptional regulation and differential distribution of FXYD1. The FXYD1 promoter contains methylated cytosines and a predicted CpG island. 5 ′aza-cytidine markedly increased FXYD1 mRNA levels. Mouse heart showed lower methylation in fxyd1 promoter and higher fxyd1 expression; while brain had a higher methylation of the fxyd1 promoter and lower fxyd1 expression (Deng et al., 2007). In brain, the fxyd1 mRNA level is lower in the frontal cortex than in cerebellum, which correlates with the more frequent promoter methylation found in frontal cortex (Banine et al., 2011). The fxyd1 promoter is a target of the Methyl CpG binding protein 2 (MeCP2), a common transcriptional modulator. MeCP2 directly bound to methylated CpG in the fxyd1 promoter and repressed fxyd1 transcription (Deng et al., 2007). On the other hand, activating histones, such as histone 3 acetylated at lysines 9 and 14 (H3K9/14ac) and histone

CONCLUSIONS
The expression of Na,K-ATPase is transcriptionally regulated by hormones, growth factors, lipid mediators and other extracellular stimuli through mediating transcription factor binding to promoter regions of Na,K-ATPase subunits. Many regulators of Na,K-ATPase are involved in the response to demands of cellular activity and ion homeostasis. Together with the spatial and temporal regulation of Na,K-ATPase subunits and their individual isoforms, this allows for a complex and controlled regulation of Na,K-ATPase in response to physiological demands in a tissue-specific manner. Interestingly, various recently identified transcriptional regulators of Na,K-ATPase are transcription factors that are also activated during development, in stem cells and during epithelial-mesenchymal transition (EMT). EMT is a switch in which epithelial cells undergo a shift from a well-differentiated polarized epithelial phenotype to a fibroblastic, mesenchymal phenotype and is a natural developmental process in tissue and organ formation. EMT can also be activated during wound healing, in tissue fibrosis, and is a major factor in cancer developing into a malignant disease (Kalluri and Weinberg, 2009). Vice versa, EMT activated transcription factors regulate the expression of Na,K-ATPase. For example, TGF-β and FGF up-regulate the expression of ZEB1, ZEB2, and Snail; steroid hormones, IGF-1 and PGE2 also induce ZEB1 and ZEB2. And ZEB1 and Snail1 regulate the transcription of Na,K-ATPase. Most recently, we identified the β 1 -subunit as a target of the Sonic hedgehog (Shh) signaling pathway (Lee et al., 2015). Shh is a critical morphogen involved in patterning of the early embryo and organogenesis, including the neural tube and limb system. The correlation between these signaling pathways and the transcriptional regulation of Na,K-ATPase is intriguing as elucidating the cross-regulation network among these factors may help in our understanding of the role of Na,K-ATPase with its pump-dependent and pump-independent functions in normal development, stem cell biology, and disease.