Functional properties of Na-K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) from which the cells originate. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na-K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation, and co-localization. Induction of FXYD2 by hypertonicity (500 mosmol/kgH2O with NaCl for 48 h or adaptation to 700 mosmol/kgH2O) correlated with downregulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed, first, dephosphorylation of FXYD1 at Ser68 (1–5 h) and, second, induction of FXYD2a and a decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mosmol/kgH2O and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na-K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiological stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK-293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na-K-ATPase, and their interchangeability underlies responses of Na-K-ATPase to cellular stress.
- cellular stress
the na-k-atpase catalyzes active efflux of Na+ and uptake of K+ ions, thus establishing and controlling ionic gradients across the plasma membrane in virtually every animal cell. It is the receptor for cardiac glycosides, drugs commonly used in the treatment of heart failure. Recent findings suggest that binding of ouabain to Na-K-ATPase, aside from a direct inhibition of the pump activity, initiates a signaling pathway that ultimately leads to changes in cell hypertrophy and proliferation.
Regulation of Na-K-ATPase occurs at several levels. It may entail regulation of gene expression of the obligatory α- and β-subunits (long-term regulation, which may serve as an adaptation response) or recruitment/internalization of the active pump units to/from plasma membrane in response to certain physiological stimuli, which can serve as an immediate regulatory response. On top of this, multilayered regulation of Na-K-ATPase includes modulation of the intrinsic properties of the enzyme via physical association with the so-called FXYD proteins (reviewed in Refs. 29, 31). This is a family of small (6.5–17 kDa) single-span membrane proteins with structural homology (61). The family includes phospholemman (PLM) (FXYD1), the major plasma membrane substrate for PKA and PKC phosphorylation in heart (49); the γ-subunit (FXYD2) (45), which is expressed primarily in kidney in at least two splice variants (6); Mat 8 (FXYD3), a gene upregulated in epithelial cells by oncogenic Ras and Neu and expressed in human breast and prostate tumors (46); corticosteroid hormone-induced factor (CHIF) (FXYD4) (7); dysadherin, or related to ion channel (RIC) (FXYD5) (27); FXYD6 (phosphohippolin) (61); and brain-specific FXYD7. Most of the homology is within the transmembrane segment and the signature motif PFXYD (61).
To date, six members of the family have been demonstrated to be associated with the Na-K-ATPase: FXYD1 (20, 26), FXYD2 (6, 51), FXYD3 (22), FXYD4 (10), FXYD5 (43), and FXYD7 (11). Remarkably, association of Na-K-ATPase with each of the FXYD proteins resulted in a somewhat different modulation of the kinetic properties of the Na-K-ATPase. FXYD4 (10, 30) and FXYD5 (43) stimulated Na-K-ATPase activity by either increasing the affinity for Na+ or increasing Vmax, whereas FXYD3 (22), FXYD7 (23), and FXYD2 exhibited an inhibitory effect by either decreasing the affinity for Na+ and/or K+ or reducing the Vmax of the pump (2, 42, 67). Overexpression of FXYD1 reduced apparent affinity for Na+ and K+ in heterologous expression systems (4, 20) and decreased the Vmax of the Na pump in adult rat myocytes (72). Phosphorylation of FXYD1, on the other hand, seemed to release the inhibition with a subsequent activation of the pump (13, 28).
Some of the FXYD family members exhibit a high degree of tissue specificity. FXYD1 is highly expressed in heart and brain (cerebellum and choroid plexus) (20, 26), but in kidney it is limited to the juxtaglomerular apparatus and blood vessels (68). Conversely, FXYD2 is only found in kidney in healthy animals (45). In stomach, expression of FXYD3 is in mucous cells (22), while FXYD4 is expressed in distal colon and collecting duct in kidney (18). Whether expression of FXYD6 and FXYD7 in the central nervous system overlaps is not yet known. Expression of FXYD5 in normal tissue is preferentially in kidney, intestine, spleen, and lung (43). In kidney, FXYD5 is at the basolateral membrane of connecting tubules and the intercalated cells of collecting duct, but it was also detected on apical membranes in thin limb of Henle, suggesting a potential role in addition to being part of the Na-K-ATPase complex.
Tissue/cell-specific expression of the regulatory FXYD subunits of Na-K-ATPase is not static, however, and may be changed to adapt to a given physiological or pathological situation. For instance, significant upregulation of FXYD1 was observed in response to cardiac infarction (57) and nerve injury (19). Expression of FXYD1 is also stimulated in muscle with exercise (54). Gene array studies revealed expression of FXYD2 in hippocampus in response to chemical insult (38). Expression of FXYD3 may be induced by ras- and neu-oncogenes (46), while expression of FXYD4 is regulated by corticosteroid and K+ deprivation (66). FXYD2, which is normally limited to kidney, can also be induced in cells of a variety of different origins in culture by exposure to different kinds of cellular stress (hypertonicity, heavy metals, exogenous oxidation, heat shock) (67).
Here we present evidence that expression of FXYDs is even more plastic in cells in culture, and cells of different origin may express more than one FXYD at a time (at protein and/or mRNA level). Moreover, we show that cellular stress (hypertonicity) initially caused dephosphorylation with a subsequent downregulation of endogenously expressed FXYD1 followed by its replacement with a newly synthesized FXYD2a within the Na-K-ATPase complex in NRK-52E cells. As we showed previously (67), induction of FXYD2a by 48-h exposure to hypertonicity correlated with a significant reduction of Na-K-ATPase activity. Here we observed ∼20% reduction in ATPase activity even after 5 h of hypertonic challenge. Because no FXYD2a was synthesized yet at this point (67), but dephosphorylation of FXYD1 was dramatic, we suggest that the latter event is employed as a first line of cell defense to survive an apoptotic insult under stress-related conditions. The data support the hypothesis that alterations of Na-K-ATPase properties entail exchanges of FXYD subunits, and this phenomenon represents an important adaptive mechanism. Some preliminary data have been presented (24).
MATERIALS AND METHODS
Antibodies and cell lines.
Polyclonal anti-sera K1 and K3 (60) were used for detection of the α1-subunit of Na-K-ATPase on blots. The VG4 monoclonal antibody (3) was employed for immunoprecipitation of α1. The RCT-G1 polyclonal antibody raised against the COOH-terminal peptide (5) was used to identify both splice variants of FXYD2. On blots, nonphosphorylated FXYD1 (PLM) was detected with the PLM-C2 rabbit anti-serum raised against the COOH-terminal sequence of PLM (the generous gift of Dr. J. Y. Cheung, Thomas Jefferson University, Philadelphia, PA (59). The form of FXYD1 that is phosphorylated at Ser68 was detected with the CP68 antibody provided by Dr. J. R. Moorman (University of Virginia Health System, Charlottesville, VA) (55). For immunoprecipitation of FXYD1, PLM-C1, an affinity-purified polyclonal rabbit antibody against the COOH terminus, was employed (a kind gift of Dr. L. R. Jones, Krannert Institute of Cardiology, Indiana University, Indianapolis, IN). The polyclonal antibody against FXYD5 (anti-dysadherin, ab2248) was from AbCam (Cambridge, MA).
NRK-52E, C6, MDCK, LLC-PK1, Caco-2, OK, HEK-293, HeLa, and SH-SY5Y cell lines were purchased from American Type Culture Collection and grown according to the manufacturer's recommendations. mIMCD3 cells were a kind gift of Dr. J. Capasso (University of Colorado Health Sciences Center, Denver, CO). Differentiation of SH-SY5Y cells was with retinoic acid (10 μM).
Cell culture and hypertonicity treatment.
The normal rat kidney epithelial cell line (NRK-52E) was grown to 85–90% confluency in Dulbecco's modified Eagle's medium with 10% FBS (300 mosmol/kgH2O). The medium was then replaced with either control or hyperosmotic (500 mosmol/kgH2O total) medium supplemented with NaCl. In the case of serum reduction, cells were incubated overnight in a medium containing 0.5% FBS before hypertonicity treatment in the same concentration of serum. Flasks were washed with Dulbecco's PBS with Ca2+ and Mg2+ at various times and frozen at −80°C.
Adaptation of NRK-52E cells to high hypertonicity was similar to what was described for mIMCD-3 cells (15). Briefly, hypertonicity was raised in the medium by successive increments of 50 mosmol/kgH2O (with NaCl). Cells were allowed to adapt to each new medium for 2–3 wk before the next increase in osmolality. Passaging was performed when cells reached confluency.
Membrane preparations and enzyme purification.
Crude membrane preparations were obtained from scraped cells by homogenization and differential centrifugation (5). Isolation of membranes from rat kidney outer medulla was performed as described elsewhere (60). Partial purification of Na-K-ATPase from cell membranes (1.4 mg/ml) was with SDS extraction (0.56 mg/ml) and sedimentation on 7–30% sucrose gradients. Typical specific activity of Na-K-ATPase was ∼50–150 μmol Pi·mg protein−1·h−1 in preparations from NRK-52E cells.
To obtain cell lysates, a buffer containing 50 mM Tris-Cl, pH 8.0, 5 mM EDTA, 1% NP-40, and protein phosphatase inhibitors cocktail I (Sigma) (1:100, vol/vol), was used. Cells were scraped and triturated, and insoluble material was removed by centrifugation at 3,000 g for 10 min (Sorvall, SS-34).
Na-K-ATPase activity was measured in SDS-purified preparations from either control or adapted cells as a function of Na+ concentration in media containing 3 mM Tris-ATP, 4 mM MgCl2, and 30 mM histidine, pH 7.4, and in the presence of 20 mM K+. All the reactions were performed at 37°C for 30 min with and without 3 mM ouabain, and ouabain-sensitive Pi release was measured colorimetrically by using the Fiske-Subbarow method. Data were analyzed by nonlinear regression using Sigma Plot Graph System (Jandel Scientific). Na+ activation curves were fitted according to the Hill model for ligand binding.
Activity in crude membranes was measured using the so-called “yellow method,” which tolerates more protein and lipid. After quenching of the ATPase reaction with acid and molybdenum as in the Fiske-Subbarow method, the modification involves the extraction of liberated Pi as the unreduced (yellow) phosphomolybdenum complex into an organic phase (water-saturated n-isobutanol) and reading the absorbance at 380 nm. Protein phosphatase inhibitor was added throughout the isolation of membranes and activity measurements.
Plasma membrane-enriched fractions from NRK-52E cells (1 mg/ml) were solubilized with 3 mg/ml n-dodecyl octaethylene glycol monoether detergent (C12E8; Calbiochem) (6) for 10 min at room temperature in buffer A, containing 140 mM NaCl, 25 mM imidazole, and 1 mM EDTA, pH 7.3. At the end of the incubation, the solubilized material was diluted with 2 vol of buffer A and sedimented by centrifugation for 30 min at 20,000 g at 4°C. The supernatant was incubated with primary antibodies (PLM-C1 to immunoprecipitate PLM or VG4 to immunoprecipitate the α-subunit) or control IgG (rabbit or mouse, respectively, at 1–2 μg/ml) overnight at 4°C with rocking. The immune complexes were collected after 2 h of incubation with 40 μl of secondary goat anti-rabbit or goat anti-mouse IgG antibodies covalently bound to agarose beads (Sigma). Immunoprecipitates were collected by centrifugation at 9,300 g for 10 min at 4°C and washed four times with buffer A containing 0.05% C12E8. After the final wash, the pellet was resuspended in 1× electrophoresis sample buffer.
Membrane fractions were resolved either on SDS-Tricine or on 4–12% MES-SDS gels (NuPage system, Invitrogen). Proteins were transferred to nitrocellulose and incubated with antibodies, and detection was with chemiluminescence. For positive control samples, membrane preparations from kidney or canine cardiac sarcolemma (37) were used. Quantification was with ImageQuant TL image analysis software (Amersham).
NRK-52E cells, either wildtype or adapted to 700 mosmol/kgH2O, were seeded into 38-mm2 wells of 96-well flat-bottomed plates (5 × 103/ well) in quadruplicate and allowed to adhere overnight. Cell proliferation over the following 2–6 days was assayed based on the cleavage of the tetrazolium salt WST-1 (Roche Diagnostics) by mitochondrial dehydrogenases in viable cells. Quantification was with a microtiter plate reader at 450 nm.
Immunocytochemistry was performed as described elsewhere in more detail (6). Briefly, cells were fixed with 2% periodate-lysine-paraformaldehyde solution for 30 min at room temperature, followed by 5-min incubation with 1% SDS in PBS, several washes with PBS, and blocking with 5% goat serum in PBS to prevent nonspecific binding of secondary antibodies. McK1 antibody was used for α1 detection, whereas PLM-C1 (described above) and CP68 antibodies were used to probe nonphosphorylated or phosphorylated FXYD1, respectively. The secondary antibodies were either Cy3-conjugated goat anti-mouse IgG (1:300; Accurate) or fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:300; Jackson ImmunoResearch). Slides were examined with a Nikon TE300 fluorescence microscope equipped with a Bio-Rad MRC 1024 scanning laser confocal system (version 3.2).
Total RNA from cells was prepared with the RNeasy system (Qiagen). The cDNA was obtained using 1 μg of total RNA, oligo(dT) as the priming oligonucleotide, and Super Script II RT (Invitrogen). For HEK-293 and HeLa cells, cDNA was obtained with the Super Script III Cell Direct cDNA synthesis system (Invitrogen). The PCR was performed in the presence of 3 mM MgCl2 with either Taq polymerase (Fisher) or Platinum Taq polymerase (Invitrogen). PCR products were separated by electrophoresis in 2% agarose gels. Primers were chosen according to sequences of FXYD genes from rat, mouse, and human (Table 1), using software provided by Invitrogen.
FXYD1 is associated with Na-K-ATPase in NRK-52E cells.
Expression of proteins in cultured cells often parallels the expression pattern in the tissues from which they originate. However, expression of regulatory subunits of Na-K-ATPase (FXYDs) does not entirely follow the same paradigm. FXYD1, PLM, is abundant in muscle, brain, and heart, where it represents the major substrate for PKA and PKC. In kidney, however, its expression is limited to afferent arteriole and lacis cells in the juxtaglomerular apparatus (68). Nevertheless, as shown in Fig. 1A, the FXYD1 protein was readily detectable in crude membrane preparations (P2) from renal NRK-52E cells, which possess characteristics of proximal tubule cells. Moreover, FXYD1 remained tightly bound to Na-K-ATPase from NRK-52E cells during a purification procedure that involves mild SDS treatment of crude membranes and extraction of the majority of membrane proteins other than Na-K-ATPase (5). The blots were stained with the C2 antibody that is specific for nonphosphorylated FXYD1 and exhibits a very weak cross-reactivity with phosphorylated FXYD1 (55). No signal was observed when identical blots were stained with the CP68 antibody (raised against the peptide phosphorylated at the Ser68 site) (55) (not shown). Because no protein phosphatase inhibitors were employed here, the assumption was that any phosphorylation of FXYD1 at the Ser68 site was labile and was lost during membrane preparation. Therefore, unless otherwise specified, we used staining by C2 as a measure of the total level of FXYD1 expression.
Interaction of the α-subunit of Na-K-ATPase and FXYD1 was further corroborated by their co-immunoprecipitation from crude membranes solubilized with the nonionic detergent C12E8 (Fig. 1B). Antibody against FXYD1 brought down the α-subunit of Na-K-ATPase, and anti-α antibodies precipitated FXYD1 as well (not shown). [The doublets of FXYD1 seen in some figures but not in others may have something to do with the different gel systems employed (SDS-Tricine vs. 4–12% gradient SDS-MES gels from Invitrogen) and were not investigated further.] Together, the data indicate that FXYD1 is an authentic component of Na-K-ATPase in renal NRK-52E cells.
Interestingly, the ratio between FXYD1 and the α-subunit was significantly lower in the purified preparation of Na-K-ATPase compared with crude membranes (Fig. 1A), implying the existence of a pool of FXYD1 not tightly bound to Na-K-ATPase.
We used immunocytochemistry to monitor the location of FXYD1 in NRK-52E cells. Cells were fixed with 2% periodate-lysine-paraformaldehyde and stained for FXYD1 and the α-subunit of Na-K-ATPase. Two different antibodies were employed here to distinguish between phosphorylated (Ser68) and nonphosphorylated forms of FXYD1. As shown in Fig. 2, subcellular location of FXYD1 was apparently affected by the phosphorylation status of the protein. Good co-localization of FXYD1 phosphorylated at the Ser68 site and α was observed at plasma membrane (Fig. 2, A–C), while a majority of unphosphorylated FXYD1 was seen intracellularly (Fig. 2, D–F). The data are in line with prior data (39) suggesting that phosphorylation of FXYD1 may be a prerequisite for trafficking to plasma membrane.
Thus, on the basis of immunofluorescence data, we speculate that a “free pool” of FXYD1 may consist of an intracellularly located nonphosphorylated FXYD1, whereas a major fraction of FXYD1 associated with the pump at plasma membrane consists of a phosphorylated form of the protein.
We also detected FXYD1 in other renal cell lines originating from proximal tubules: porcine LLC-PK1 and opossum OK cells (Fig. 3). Meanwhile, no FXYD1 was detected in canine MDCK (Fig. 3) or mouse mIMCD3 cells (not shown), both originating from distal nephron segments, or in embryonic HEK-293 cells. The antibody's epitope is conserved. This implies cell (tubule)-specific control of FXYD1 expression in culture. Interestingly, in kidney, proximal tubules are positive for FXYD2a, while more distal segments express either FXYD2b (distal convoluted tubules) or a combination of FXYD2b and FXYD2a (inner medullary collecting duct). Although abundant in tissue, FXYD2 is normally absent from any mammalian renal cell line (5, 62). The attractive hypothesis is that FXYD1 substitutes for FXYD2a within the Na-K-ATPase complex in cells in culture to adjust intrinsic properties of the pump properly for the environment.
Transfection with FXYD2 affects the expression of endogenous FXYD1.
We have shown previously that expression of the FXYD2 splice variants can be achieved in NRK-52E cells by transfection (2, 5), although the level of expression of FXYD2a or FXYD2b was never as high as in kidney membranes. With both FXYD2a and FXYD2b, there was a reduction in the activity of Na-K-ATPase and a reduction in the rate of cell growth. Because FXYD1 is endogenously expressed in NRK-52E cells as an authentic component of Na-K-ATPase, we tested whether expression of FXYD2 by transfection would cause a replacement of one FXYD protein with another. Several clones expressing splice variants of γ (FXYD2) have been tested, and Fig. 4 shows a representative Western blot analysis of crude membranes from γa-, γb-, and mock-transfected cells. Transfection with γa correlated with only a slight (if any) reduction of the total level of FXYD1 expressed in NRK-52E: 92 ± 13% (n = 3) expressed as the ratio between the staining of PLM (C2) and the α-subunit (K3) in transfected vs. control cells. In contrast, the FXYD1/α ratio was significantly lower in γb-transfectants compared with γa- (18.1 ± 4.5%, n = 4) and mock-transfected cells (11.2 ± 2.3%, n = 4), even though the amount of γ was lower in γb- compared with γa-transfectants (Fig. 4). The data imply a cross talk in the regulation of FXYD protein expression and a potential competition between γb and PLM for binding to Na-K-ATPase. Because both γa- and γb-transfectants exhibited similar slow growth compared with mock-transfected cells, the conclusion is that introduction of FXYD2 rather than relative changes in expression of FXYD1 had the impact on the rate of cell proliferation, which is in agreement with our recent data on knockdown of FXYD2 in NRK-52E cells (67), where silencing restored Na-K-ATPase activity and the normal rate of growth in the continued presence of hypertonicity.
Reciprocal regulation of FXYD1/FXYD2a expression under stress-related conditions.
As we demonstrated previously (67), FXYD2a is not expressed in renal cells in culture under normal conditions but can be induced by several kinds of cellular stress, such as hypertonicity (with NaCl and sucrose, but not with urea), heat shock, exogenous oxidants, or heavy metal treatment. The newly synthesized FXYD2a associates with the Na-K-ATPase and inhibits its activity. Here we tested what happens with the constitutively expressed FXYD1 during stress-mediated induction of FXYD2a.
Figure 5 shows a representative Western blot of crude membranes from either NRK-52E cells (Fig. 5A) or C6 glioma cells (Fig. 5B) exposed to isotonic (control) or hypertonic conditions (500 mosmol/kgH2O with NaCl) in the presence of 10% FBS. In both cases, induction of FXYD2a correlated with a reduction of the total level of FXYD1. (No protein phosphatase inhibitors were added during membrane preparation.) On the basis of densitometry analysis, the FXYD1/α ratio was decreased by 31.8 ± 3.5% (n = 5) in membranes from NRK-52E cells acutely treated with hypertonicity compared with control. Downregulation of FXYD1 with hypertonicity was greater (at least 50% or more) in C6 cells, suggesting specificity of cellular response.
The changes in the reciprocal expression of FXYD1/FXYD2a were even more dramatic in NRK-52E cells slowly adapted to hypertonicity. Although these cells originate from proximal tubules (40), which do not experience high hypertonicity in vivo, NRK-52E can be adapted to live under high salt, similar to what was described for mIMCD3 cells (15). Adapted cells (700 mosmol/kgH2O with NaCl in the presence of 10% FBS) are viable but proliferate much slower than control cells grown in 300 mosmol/kgH2O medium (Fig. 6A). Western blot analysis of crude membranes showed that adaptation to high salt caused 1) an increase in protein expression of the α-subunit (1.7 ± 0.5-fold, n = 4) and 2) a reciprocal switch between FXYD proteins: induction of FXYD2a was accompanied by a complete loss of FXYD1 from cells slowly adapted to high salt (Fig. 6B). Functionally, there was a significant (1.5 ± 0.4-fold, n = 3) increase in Na-K-ATPase activity in membranes from adapted cells that correlated nicely with an increase in the amount of α, implying no significant changes in activity per unit of protein measured under optimal conditions. The data are in qualitative agreement with a report by Capasso et al. (16) that showed a 5-fold upregulation of α- and β-subunits in mIMCD3 cells adapted to live under hypertonic conditions (600 mosmol/kgH2O with NaCl) as well as a 10-fold or larger increase in Na-K-ATPase activity in cell lysates.
The complete substitution of one FXYD for the other had a dramatic effect on Na-K-ATPase kinetic properties. As shown in Fig. 6C, the apparent affinity for Na+ was significantly decreased in preparations of Na-K-ATPase isolated from adapted cells where FXYD1 was completely replaced with FXYD2a. On the basis of nonlinear regression analysis of data fit to the Hill equation, substituting K0.5 (half maximal apparent affinity) for Kd (dissociation constant), K0.5 for Na+ was shifted from 4.9 ± 0.6 mM (n = 6) in wildtype NRK-52E cells (expressing “pure” FXYD1) to 12.6 ± 0.8 mM (n = 5) in adapted cells (expressing pure FXYD2a). The data show an even greater K0.5 for Na+ than measured in partially purified enzyme from rat renal medulla (9.5 ± 0.4 mM) (2) or whole mouse kidney (7.6 mM) (36), where there is a mixture of γa and γb. The measured affinity is within the physiological range of the intracellular concentration of Na+ (14.9 ± 0.6 mM) determined in proximal tubules (9). Thus the data highlight the physiological significance of FXYD2a as a modulator of apparent affinity for Na+ of Na-K-ATPase.
Another example of reciprocal changes in FXYD1/FXYD2 expression under stress conditions is shown in Fig. 7. NRK-52E cells were treated with 1 mM ouabain for 24 or 96 h. Although this concentration of ouabain should inhibit the rat Na-K-ATPase ∼85–90%, cells were not dying and only slowed down in the rate of proliferation (not shown). This is very different from HeLa, where ouabain induces apoptosis (53). On the basis of Western blot analysis, there was induction of FXYD2a in a time-dependent manner. Staining of an identical blot with the C2 antibody revealed a significant reduction in the level of nonphosphorylated FXYD1, while no signal was detected with the CP68 antibody (not shown). Because no precautions were taken to keep phosphorylation at Ser68 intact, the data suggest a reduction in the total level of FXYD1 during ouabain treatment while gaining FXYD2a. Reduction in the FXYD1/α ratio was already significant after 24 h of treatment (66.8 ± 7.0%, n = 4) and was even more pronounced with longer exposure (96 h) (81.7 ± 4.3%, n = 3). At this point it is not clear why ouabain-treated cells induce expression of FXYD2a (which acts as an inhibitor of Na-K-ATPase) when the pump is already inhibited by the ouabain. One of the possibilities is that ouabain activates cell signaling pathways convergent with those employed for upregulation of FXYD2a by stress.
All together, the data suggest that interchangeability of FXYDs, regulatory subunits of Na-K-ATPase, represents a part of a general mechanism to provide a proper cellular response to physiological or pathological stimuli.
Dual response of FXYD1 to hypertonicity and trophic factor-dependent pathways.
Hypertonicity activates multiple signaling pathways entailing protein kinases and protein phosphatases. To monitor whether phosphorylation of FXYD1 is changed in response to this kind of stress, NRK-52E cells were subjected to hypertonicity (500 mosmol/kgH2O with NaCl for 48 h) in the presence of 10 or 0.5% FBS, and cell lysates were tested on Western blots (Fig. 8). The reduced serum condition is a method often used experimentally to amplify the effects of hypertonicity on signaling pathways. Protein phosphatase inhibitor cocktail was added to the lysis buffer to preserve phosphorylation of FXYD1. In both serum concentrations, there was induction of FXYD2a with no substantial changes in the level of α1 of Na-K-ATPase (Fig. 8A). However, the response of FXYD1 was different depending on serum concentration. In normal osmolarity, there was a higher proportion of phosphorylation of FXYD1 in the presence of 10% serum than 0.5% serum. A hypertonic challenge of 24–48 h in the presence of 10% FBS caused a reduction in the level of the nonphosphorylated form of FXYD1: the FXYD1/α ratio was decreased in the stressed cells by 31.4 ± 5.6% (n = 3). Simultaneously, there was an even more significant drop (>60%) in the level of the phosphorylated form of FXYD1. The data imply a decrease of the total level of the FXYD1 protein as well as lability of phosphorylation under stress conditions (Fig. 8A).
Interestingly, reduction in the phosphorylation level seen in 10% serum was observed even after short-term (up to 5 h) exposure to hypertonicity (Fig. 8B). To test whether dephosphorylation of FXYD1 is functionally significant, NRK-52E cells were treated with hypertonicity (500 mosmol/kgH2O with NaCl) for 5 h, and crude membranes were isolated and analyzed in the presence of protein phosphatase inhibitor. There was a reduction of Na-K-ATPase activity by 23.3 ± 7.1% (n = 3) in membranes from stressed cells compared with control. Less than 5% of the activity was lost in cells exposed to similar conditions for just 1 h. We previously showed that induction of FXYD2a by 48-h exposure to hypertonicity correlated with a significant reduction of Na-K-ATPase activity. Since no FXYD2a was synthesized yet after 5 h in hypertonic medium (67), we suggest that dephosphorylation of FXYD1 led to inhibition of the Na pump activity to survive an apoptotic insult under stress-related conditions.
The incubation of NRK-52E cells in reduced serum isotonic medium resulted in a significant loss of phosphorylation of FXYD1 at Ser68 (>50% compared with phosphorylation of FXYD1 in 10% serum-supplemented isotonic conditions) that was not significantly affected further by application of hypertonicity (Fig. 8A). However, the reduction in the level of nonphosphorylated FXYD1 under hypertonic conditions in the presence of 0.5% FBS was more dramatic than in serum-supplemented medium: the FXYD1/α ratio was reduced by >85% (observed in 3 independent experiments).
Serum growth factors apparently enhance pathways that result in higher basal phosphorylation of FXYD1. Dephosphorylation of FXYD1 is the major event accompanying hypertonic stress in NRK-52E cells grown in 10% serum, whereas significant downregulation of nonphosphorylated FXYD1 occurred under hypertonic stress in the reduced serum conditions. Interestingly, cells grown in low serum proliferated significantly slower than control cells grown in the presence of 10% FBS and at a rate similar to those grown in serum-supplemented conditions but treated with hypertonicity (Fig. 8C). Thus there was an apparent correlation between the rate of proliferation and the phosphorylation of FXYD1. Since phosphorylation of FXYD1 has been implicated in activation (or release of inhibition) of Na-K-ATPase, this is further supporting evidence that Na pump activity is critical in supporting cell growth.
Stress-induced changes in FXYD1/FXYD2 entail regulation of gene expression.
Reduction in the level of FXYD1 protein may indicate that a transcriptional or translational block occurred in response to hypertonicity, or that mRNA levels are normal and degradation was increased. To detect any change in mRNA level, we performed RT-PCR analysis for FXYD1 and FXYD2a from NRK-52E cells grown under normal conditions or in hypertonic medium (acute treatment for 48 h at 500 mosmol/kgH2O with NaCl or chronic treatment with 700 mosmol/kgH2O). To our surprise, we detected the PCR product corresponding to the FXYD2a mRNA under all experimental conditions, including the controls, although the intensity of the band was higher with acute hypertonicity and most pronounced under chronic hypertonicity (Fig. 9). In contrast, the intensity of the FXYD1 PCR product bands declined with acute hypertonic treatment and was completely lost in the adapted cells. The data suggest that 1) there is a transcriptional and a translational block in expression of FXYD2a in NRK-52E under normal tissue culture conditions, in line with related observations of Capasso et al. (17) on inner medullar collecting duct cells, and 2) that FXYD1 mRNA levels are regulated by hypertonicity. Whether the latter is due to the rate of transcription of the FXYD1 gene or to changes in stability of the FXYD1 mRNA awaits further investigation.
Expression of FXYDs in different cell lines.
The finding of FXYD1 as an authentic component of Na-K-ATPase in renal cells was quite unexpected. Therefore, we performed a systematic RT-PCR analysis of FXYD expression in several cell lines. Positive controls for the specific primers were included for each species. Table 2 summarizes the data from NRK-52E, mIMCD-3, and the Caco-2 intestinal epithelial cells lines grown under normal conditions or exposed to hypertonicity (500 mosmol/kgH2O with NaCl for 48 h). Interestingly, all of these cell lines express FXYD3 and FXYD5 mRNAs under basal conditions, the level of which was unaffected by hypertonicity in NRK-52E and mIMCD3 cells but reduced significantly in Caco-2 cells. In contrast, all cells demonstrated an increase in the expression of FXYD2a mRNA in response to high salt treatment. Only mIMCD3 cells showed an increase in the expression level of FXYD2b mRNA. NRK-52E cells were the only ones that expressed FXYD1 mRNA, and it showed downregulation under hypertonic conditions.
Similar PCR analysis was performed on human HEK-293, HeLa, and retinoic acid-differentiated SH-SY5Y cells grown under isotonic conditions (300 mosmol/kgH2O). As can be seen from Table 3, FXYD5 was detected in all three cell lines, whereas FXYD3 was identified in HeLa and FXYD6 was found in HEK-293. Surprisingly, human neuroblastoma cells (SH-SY5Y) displayed almost a complete set of FXYDs except FXYD2a and FXYD1. Expression of FXYD6 mRNA was the most pronounced, although “kidney-specific” FXYD2b and FXYD4 were also found in these “neuronal” cells. The data further support the hypothesis of plasticity and complexity of FXYD expression in different settings such as tissues or cultured cells.
Because HEK-293 and HeLa cells have been used in the past as expression systems for different FXYDs, we have tested whether FXYD5 was expressed as a protein in these cells. Figure 10 demonstrates the results of Western blot with both crude membranes and partially purified preparations of Na-K-ATPase from HEK-293 and HeLa cells. The major bands recognized by anti-dysadherin antibody were about 17–20 kDa (marked with a single arrow), which very likely represented the core protein (with a calculated molecular mass about 17 kDa), and a fuzzy band within a range of 28–35 kDa that is presumably glycosylated (marked with a double arrow). We detected more highly glycosylated FXYD5 (50 kDa) in membranes from LLC-PK1 cells (not shown). FXYD5 is a heavily O-glycosylated protein (65), and multiple patterns of glycosylation of FXYD5 are expected that may vary in different cell types. At least some of the FXYD5 remained bound to Na-K-ATPase during the SDS extraction purification procedure, consistent with the idea that that FXYD5 represents an endogenous regulatory subunit of Na-K-ATPase expressed in these cells.
It is been several years since the discovery of the FXYD family members (61) as tissue-specific regulators of Na-K-ATPase (reviewed in Ref. 21). FXYD2, the γ-subunit, has been studied the most extensively, and the fact that this protein is complexed with the pump in several nephron segments in the kidney but not in renal cells in culture (5, 62) supported the hypothesis that FXYD2 (and FXYDs in general) are not obligatory but rather auxiliary subunits required for fine tuning the pump properties.
The novelty of this work is a demonstration of FXYDs as essential subunits of the Na-K-ATPase complex, and a demonstration of their interchangeability. There are at least two lines of evidence in support. First, every cell line we have checked apparently contains one or more FXYD mRNA. Remarkably, there is no direct correlation between FXYDs expressed in a tissue of origin and a cell line. For instance, expression of PLM (FXYD1) in kidney is limited to afferent arterioles and lacis cells in the juxtaglomerular apparatus (68). Nevertheless, it is highly expressed in cultured renal cells of proximal origin, NRK-52E, LLC-PK1, and OK cells (Fig. 1), but not in MDCK and mIMCD3 cells derived from more distal tubules. On the other hand, kidney is almost the only tissue expressing FXYD2 under normal conditions (45), and yet no protein can be detected in renal cells in culture (5, 62, 64). Expression of FXYD5 in kidney is restricted to distal nephron, connecting tubules, collecting tubules, and intercalated cells of the collecting duct, although labeling of the apical membrane in long thin limb of Henle's loop was also observed (43). Here we found a message for FXYD5 in renal cell lines of different origin: NRK-52E (proximal tubules), IMCD3 (collecting duct), and HEK-293 cells. Moreover, we found FXYD5 protein in HEK-293 and HeLa cells. This may be related to cell transformation, though, since FXYD5 was identified in different kinds of tumors (reviewed in Ref. 33). A similar argument may explain expression of FXYD3 mRNA in a majority of cell lines tested in this work (NRK-52E, mIMCD3, Caco-2, HeLa cells). FXYD3 was originally identified in breast and prostate tumors, and expression of FXYD3 may be induced in culture by ras- and neu-oncogenes. Whether expression of FXYD3 and FXYD5 proteins parallels the expression of FXYD3 and FXYD5 mRNAs in cultured cells remains to be investigated.
Second, reciprocal exchange of FXYD1/FXYD2 proteins has been observed with stimuli such as hypertonicity or ouabain treatment. Interchangeability is apparently required for proper adaptation of cells to any given physiological or pathological condition.
The major implication of this work is that Na-K-ATPase in cells in culture is always paired with a FXYD protein(s), the expression of which does not necessarily parallel the expression pattern in the tissue(s) from which the cells originate. One of the outcomes is a call for rigorous systematic analysis of kinetic influences of the FXYD proteins on Na-K-ATPase properties isolated from the dynamic background of host cell FXYDs. For instance, the functional significance of FXYD2 for the Na-K-ATPase complex was earlier assessed in different heterologous expression systems. There was a general agreement that a major functional consequence of association with FXYD2 is a modulation (reduction) of apparent affinity for Na+. Indeed, introduction of FXYD2 into the Na-K-ATPase complex resulted in an ∼1.5-fold decrease in the affinity for Na+, from 5 to 7.5 mM (reviewed in Ref. 4). However, K0.5 Na+ was still lower than in purified enzyme from kidney (8.4–9.5 mM), and therefore skepticism was raised concerning the physiological role of FXYD2. Here we showed that apparent affinity for Na+ can be indeed modulated by association with FXYD2a and reduced to values (12.5 mM) even exceeding those found in purified kidney enzyme, but only in homogenous preparations of Na-K-ATPase containing FXYD2a and no FXYD1 as regulatory subunit. Such a change would be large in a physiological context, bringing the affinity close to the intracellular concentration of 14.9 mM.
Aside from modulation of the affinity for Na+, other functional effects of FXYD2 were observed in heterologous expression systems including modulation of apparent affinity for K+ (2, 12), affinity for ATP (52, 63), and changes in K+ antagonism of apparent Na+ affinity (2, 63). Results in different studies were sometimes at odds. Although such discrepancies may yet depend on which splice form of FXYD2 was expressed and whether posttranslational modification occurred, it is also likely that there was an interplay between the FXYD2 introduced by transfection and the different endogenous FXYDs (Tables 2 and 3) in the Na-K-ATPase complex.
Xenopus oocytes have been used as an expression system for different FXYDs (reviewed in Ref. 31) and FXYD2 in particular (12). We have searched the expressed sequence tag (EST) database for Xenopus with tBLASTn analysis using the sequence of mouse FXYD3. Previously unidentified orthologs were found in four different libraries constructed from unfertilized egg of Xenopus tropicalis (7 entries in 3 libraries) and X. laevis (4 entries from 1 library). Protein sequences are quite similar to each other (88% identity) and probably are the products of the orthologous gene. They also possess a similar degree of homology to both FXYD3 (53 and 54% for X. tropicalis and X. laevis, respectively) and FXYD4 (46% for both) and thus could represent the orthologs of either FXYD3 or FXYD4 expressed in Xenopus egg, or could be independent FXYD proteins. If expressed as proteins, these endogenous FXYDs may have been in competition with the products of introduced FXYD cRNAs in oocyte expression studies. FXYD3 and FXYD4 from mouse show 55% identity with each other. Figure 11 demonstrates the sequence alignment (Fig. 11A) and a cladogram tree (Fig. 11B), showing phylogeny without indication of the amount of evolutionary “time.” Interestingly, the FXYD orthologs found in Xenopus egg libraries are only expressed in reproductive tissues (egg, testis) or tissues from early developmental stages of Xenopus embryo (gastrula, early neurula, anterior neuroectoderm, etc.) and nowhere else.
We also showed that cells of different origin may express more than one mRNA at a time, suggesting that more than one regulatory subunit of Na-K-ATPase may be expressed. Since only one molecule of any kind of FXYD can apparently be bound to α-β monomer (32), this could lead to the co-existence of different multisubunit complexes of Na-K-ATPase. This kind of phenomenon was already observed in the middle segment of inner medullary collecting duct in rats (50): all principal cells exhibited basolateral staining for FXYD2a, FXYD2b, and FXYD4. Another known example is co-expression of FXYD2a and FXYD2b in medullary thick ascending limb, demonstrated by immunocytochemistry and co-immunoprecipitation of mixed oligomeric complexes (6).
Phosphorylation of FXYD1 as a mode of regulation of Na-K-ATPase.
Different functional effects of FXYD1 on Na-K-ATPase have been reported. Expression in oocytes led to a reduction of the apparent affinity for Na+, i.e., inhibition of Na-K-ATPase activity at physiological Na+ concentrations (20). Overexpression of FXYD1 in normal adult rat myocytes resulted in a significant reduction of Vmax without appreciable changes in K0.5 for Na+ and K+ (72). On the other hand, a stimulatory role for FXYD1 was proposed based on reduction of total Na-K-ATPase activity in the FXYD1−/− mouse relative to littermate controls (35). Recent findings suggest that not only expression but also phosphorylation of FXYD1 is essential for modulation of Na-K-ATPase (28, 58). Substantial activation of Na-K-ATPase has been observed in isolated sarcolemma under ischemic conditions that correlated with activation of PKA and phosphorylation of FXYD1 but not of the α-subunit (28). Treatment of isolated guinea pig cardiomyocytes with forskolin led to activation of Na-K-ATPase correlated with phosphorylation of FXYD1 at Ser68 (58). An increase in the hydrolytic activity of Na-K-ATPase and phosphorylation of FXYD10 (PLMS) was reported for shark rectal gland membranes phosphorylated by PKC (44). The opposite functional effects of FXYD1 on modulation of apparent affinity for Na+ when expressed with α/β-complex in Pichia pastoris (decrease in K0.5 of Na+) and HeLa cells (increase in K0.5 of Na+) were attributed to the presence or absence of endogenous phosphorylation at the Ser68 site, respectively (41).
Here, using phosphospecific antibodies, we showed a dramatic reduction in phosphorylation of FXYD1 per unit of protein in response to acute hypertonicity in serum-supplemented conditions. Dephosphorylation occurred within the first hour of treatment and correlated with a reduction of Na-K-ATPase activity, thus serving as an immediate response to reduce the activity of the pump while a “true repressor,” the FXYD2a subunit, is synthesized (67). On the other hand, we observed a reduction in the amount of FXYD1 along with an increase in phosphorylation of FXYD1 per unit of protein under hypertonic stress in reduced serum. In this case, as we reported previously (67) and in agreement with reports from other laboratories (14, 25, 48, 70), the activity of Na-K-ATPase is augmented. While the reduced levels of FXYD1 would imply less pump activity, the enhanced phosphorylation of FXYD1 would counteract the expected decrease in pump activity by stimulating the remaining pumps. The data suggest a consistent underlying molecular mechanism, but experimental conditions (serum levels) influence the cellular response. Interestingly, a significant drop in the expression level of FXYD1 was observed in failing rabbit and human heart samples, whereas the fraction of FXYD1 phosphorylated at Ser68 was increased dramatically (13).
On the basis of immunofluorescence, the intracellular distribution of FXYD1 depends on the phosphorylation status of the protein. While phosphorylated FXYD1 is co-localized with the α-subunit of Na-K-ATPase at the plasma membrane, the nonphosphorylated form of FXYD1 is largely retained in intracellular compartments. An intracellular location of FXYD1 may indicate a role distinct from regulation of Na-K-ATPase, a hypothesis that needs further investigation. It should be mentioned that at least FXYD1 and FXYD4 have been implicated already in the modulation of two other ion transporting systems, Na+/Ca2+ exchanger in cardiac myocytes (1, 71) and depolarization-activated K+ channels (KCNQ1) (34), respectively. On the other hand, phosphorylation of FXYD1 was required for plasma membrane delivery in MDCK cells transfected with FXYD1 constructs (39). This was consistent with an earlier report by Mounsey et al. (47) that co-expression of PKA and FXYD1 in Xenopus oocytes increased the amplitude of an ion current and the amount of FXYD1 at the plasma membrane.
In summary, we demonstrated for the first time that the cellular response to stress entails an exchange of FXYD proteins, apparently to make a smooth transition of properties of Na-K-ATPase, and this phenomenon represents a part of the adaptive mechanism. Whether an exchange in regulatory subunits is essential only for modulation of enzymatic activity or is also crucial for signal transduction pathways that involve Na-K-ATPase (8, 56, 69) awaits further investigation.
This work was supported by National Institutes of Health Research Grants HL-036271 and NS-45083 (K. J. Sweadner), DK-443351 (Massachusetts General Hospital-Harvard Medical School Center for the Study of Inflammatory Bowel Disease, E. Arystarkhova), and G12-RR-03051 and G11-HD-046326 (S. C. Specht).
We thank J. Pascoa and N. Asinovski for superb technical assistance.
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- Copyright © 2007 the American Physiological Society