Selected antibodies against the α1, α2 and α3 isoforms (α6F, McB2 and XVIF9G10 monoclonal antibodies) were characterized by western blotting to confirm their cross-reactivity with their respective protein targets in human brain and skeletal muscle (Figure 1).

The expression of Na⁺, K⁺-ATPase isoforms in plasma membrane enriched microsomes of C-20/A4 cells was investigated by western blotting. Membrane proteins were separated by SDS-PAGE in 4–12% NuPAGE gradient gels (Figure 2A). Membrane proteins were then blotted onto nitrocellulose membranes and blots incubated with isoform specific antibodies. Western blot analysis clearly established the presence of α1, α3, β1 and β2. Antibodies to α1 and α3 satisfactorily cross-reacted with protein bands between the 97.4 and 116 kDa markers (Figure 2B). As expected, the ubiquitous α1 isoform was found in higher quantities compared to α3 on the alkaline phosphatase-conjugated antibody-probed membranes. However, there was no evidence for α2 expression in chondrocytes. The western blots produced detectable bands within 10–15 min suggesting a low abundance of plasma membrane Na⁺, K⁺-ATPase in chondrocyte-like cells. The high specificity of the antibodies for their respective antigens in brain homogenates has previously been demonstrated using western blotting [25,26,30]. Due to the lack of specific antibodies to human α4 and β3, the expression of these isoforms was not investigated at the protein level.

FACS analysis demonstrated a relatively high level of α1 expression (67% positive cells) compared to α2 and α3 (1.14% and 5.28%, respectively; Table 1) in human chondrocyte-like cells. Autofluorescence was negligible, as was binding of non-immune mouse serum (Figure 2C). Expression levels of β1 and β2 isoforms were also found to be relatively high in these cells (79.49% and 87.19% respectively; see Table 1). Overall, the immunohistochemical, immunofluorescence, FACS and Western blot results presented in this paper suggest that although the α1, β1 and β2 isoforms are the most abundant isoform proteins expressed, lower, but nevertheless detectable levels of α3 protein present in human chondrocyte-like cells.

Immunocytochemical staining confirmed the presence of α1 and lower levels of α3 (Figures 3 and 4). The α1 isoform was found in abundance as expected; the monoclonal antibodies α620 and α6F, which had both reacted strongly on Western blots, also worked well in immunofluorescence experiments. The β1 and β2 subunit isoforms were also detected in roughly equal proportions (Figures 3 and 4). Immunoreactive cells exhibited strong alkaline phosphatase staining when probed with the pan α monoclonal antibody (mAb 9A7) and the α1 specific antibody; no α2 staining was observed but the α3 antibodies produced some immunostaining (Figures 3 and 4). Conventional immunofluorescence micrographs revealed strong expression of α1 on the plasma membrane. The pan α monoclonal antibody (mAb 9A7) produced the most intense immunofluorescence staining possibly due to the combined signal from α1 and α3 isoforms (Figure 3).

Recent studies of isolated bovine chondrocytes in our laboratory have revealed a high plasma membrane Na⁺, K⁺-ATPase density for these relatively small cells [25]. In addition to plasma membrane Na⁺, K⁺-ATPase, a large component is localized in intracellular membranes [32]. In bovine cartilage, the density of Na⁺, K⁺-ATPase correlates with matrix glycosaminoglycan concentrations and hence with tissue Na⁺ [27] suggesting a feedback mechanism for the regulation of Na⁺, K⁺-ATPase abundance by matrix proteoglycans. Molecular studies in bovine chondrocytes have revealed two α (α1 and α3) and two β (β1 and β2) Na⁺, K⁺-ATPase isoforms [25]. These early studies suffered from a number of limitations. First, they were completed before the cloning and identification of the α4 and β3 isoforms. Second, some of the antibodies used in the early studies were obtained from commercial sources and consequently were not sufficiently well characterized. Recent studies of Na⁺, K⁺-ATPase isoform expression in articular chondrocytes in human cartilage explants have resulted in a more consistent, but significantly more complicated description of isoform composition; 3 α (α1, α2 and α3) and 2 β isoforms (β1 and β2) are expressed in addition to several other key Na⁺ dependent transport systems [29,33].

The evidence for α3 expression in cartilage has gained considerable support since it was first proposed [25,26]. The well-characterized α3 specific monoclonal antibody XVIF9-G10 has been used successfully to demonstrate α3 expression in brain and heart [30] and articular cartilage [25,26,29]. Another well-characterized monoclonal antibody, McBX3, has been used successfully to demonstrate α3 expression in human articular cartilage [29]. There is evidence that the α3 subunit is post-translationally modified at the site of McBX3 binding in several tissues and this modification abolishes the affinity of McBX3 for its unique epitope on the α3 protein [34]. The fact that McBX3 is able to bind α3 in cartilage clearly validates that the α3 isoform expressed in cartilage is similar to the neuronal α3 isoform [29].

The expression of α3 may be related to the unusual ionic environment of chondrocytes [3]. In terms of their affinity for Na⁺, the α isoforms exhibit significantly different kinetic properties; the affinity of α3 for intracellular Na⁺ is orders of magnitude lower than α1 isoform (reviewed in [14,15]). It is therefore possible that chondrocyte Na⁺, K⁺-ATPase isozymes containing α3 subunits fulfill the vital physiological function of controlling intracellular Na⁺ during joint loading or other scenarios in which rapid changes occur to the intracellular Na⁺:K⁺ ratio [3]. Recent observations in isolated chondrocytes suggest that the α3 subunit may be preferentially upregulated along with the α1 subunit following long-term changes in extracellular Na⁺ resulting in steeper transmembrane Na⁺ gradients [32]. Chronic exposure of chondrocytes to high extracellular Na⁺ results in accumulation of α1 and α3 isoforms on the plasma membrane of chondrocytes and also in subcellular compartments [28]. Thus α3 with its low affinity for Na⁺ working in parallel with α1 isoform may allow chondrocytes to respond more effectively to physiological changes in intracellular Na⁺ that occur in vivo under load. Furthermore, the high affinity of α3 for ouabain raises the possibility of independent regulation of Na⁺, K⁺-ATPase isozymes by endogenous ouabain-like compounds.

The apparent lack of α2 expression in human chondrocyte-like cells is worthy of further discussion. Studies on isolated bovine articular chondrocytes have confirmed the absence of α2 expression [25,26]. Indeed, the predominant α isoforms expressed in isolated chondrocytes are α1 and α3, although α1 appears to be more abundant than α3 [25,26]. However, bovine and human articular chondrocytes in situ appear to express the α2 isoform [25,26,29]. The observation that α2 immunoreactivity is lost in isolated cells suggests that expression of this isoform is under hormonal regulation in cartilage. The lack of α2 in human chondrocyte-like cells supports this notion. In adipocytes (close relatives of chondrocytes) the α2 isoform is highly insulin sensitive in terms of its expression, regulation, function and cell surface recruitment [35,36]. It is possible that expression of α2 and possibly also other α isoforms is under hormonal control by insulin or IGF-1 (insulin like growth factor), a dominant anabolic mediator in cartilage.

The existence and variability of β isoforms in human chondrocytes also raises important issues that need to be addressed. Abundant expression of β1 in chondrocytes is not surprising given its ubiquitous tissue expression [20,21]. However, expression of β2 and β3 isoforms in chondrocytes, in addition to the β1 isoform, was surprising. The β2 isoform, also known as AMOG (adhesion molecule on glia) [37], is expressed in a number of tissues but mainly in neural tissues [38]. The β2 subunit is important for the development of the central nervous system [37,39,40] and mice deficient for AMOG/β2 exhibit neuronal [41].

The β3 isoform also appears to be expressed in human chondrocytes in situ [29] and human chondrocyte-like cells (this study). The β isoforms are glycoproteins displaying branched oligosaccharides to the extracellular environment. Since glycoproteins have been implicated in cell recognition and cell signaling, it may be possible that multiple β isoforms allow chondrocytes to detect changes in their extracellular environment. The α subunit of Na⁺, K⁺-ATPase has long been known to bind various intracellular cytoskeletal proteins including ankyrin [42,43] and actin [44,45]. β subunit isoforms may play a role in the biogenesis of chondrocyte Na⁺, K⁺-ATPase or modulating the longevity and intracellular trafficking of Na⁺, K⁺-ATPase complexes [14].

Chemicals were purchased from Sigma/Aldrich (Poole, UK). Chemicals used in molecular biology experiments were molecular biology grade and those used in cell culture and gel electrophoresis/ Western blotting procedures were tissue culture and electrophoresis grade respectively. Fetal bovine serum was obtained from Life Sciences (Paisley, Scotland) and Sigma/Aldrich. Molecular biology kits were obtained from BioGene Ltd (Kimbolton, UK) and Advanced Biotechnologies (Epsom, UK). Electrophoresis reagents were obtained from Novex (San Diego, USA). Oligonucleotide primers were obtained from Genosys Biotechnologies (Cambridgeshire, UK) and Genset SA Europe (Paris, France) affiliated with Helena Biosciences (Sunderland, UK). Thin-walled PCR tubes were purchased from Perkin Elmer (Norwalk, USA).

Samples of human brain and skeletal muscle were used as positive control tissues for western blot characterization of the monoclonal antibodies to the α1, α2, α3 isoforms of the Na⁺, K⁺-ATPase. Tissues were obtained from the University Hospital of Universidad de La Laguna with local Ethics Committee approval. Plasma membrane enriched microsomes were prepared as described for C-20/A4 cells below.

Well characterized non-commercial monoclonal and polyclonal antibodies to the α1, α2, α3, β1 and β2 subunits of the Na⁺, K⁺-ATPase were selected based on our previous observations and information available in the literature. The overriding priority in our selection procedure was suitability for use in human cells. The antibodies employed in this study and information and references pertaining to their specificity are shown in Table 2. Specific antibodies are currently available against three α (α1, α2 and α3) and two β isoforms (β1 and β2). Although polyclonal antibodies against the β3 subunit are also available, these are not suitable for studies on human cells as they only recognize the β3 subunit of rat and mouse [46]. Secondary antibodies used were previously pre-adsorbed with human serum proteins by the manufacturer. Rhodamine-conjugated anti-rabbit IgG (H + L) pre-adsorbed with human, mouse and rat serum proteins were purchased from Jackson ImmunoResearch laboratories Inc (PA, USA). Fluorescein-conjugated anti-mouse IgG was obtained from Dako Ltd., (Cambridge, UK). Alkaline phosphatase-conjugated anti-mouse and anti-rabbit-IgG was obtained from Sigma/Aldrich.

For all the experiments described a human chondrocyte-like cell line designated C-20/A4 was used [55]. The cells were grown in 75 cm³ flasks in Dulbecco’s modified Eagle’s medium with 1000 mg glucose/L, l-glutamine, NaHCO₃ and pyridoxine HCl (Sigma D-6046) supplemented with 5% fetal calf serum (Gibco BRL 10108-157 or Sigma F-2442) and 1% antibiotic/antimycotic solution (Sigma A-5955) in a humidified incubator (95% air, 5% CO₂). Cells from passages 12–24 were used in this study. In all experiments described, the cells were used when 95% confluent and 95% viable as determined by Trypan blue dye exclusion assays. The cells used in some immunological experiments were grown on poly-l-lysine coated slides. The morphology of the cells and the viability was not affected.

C-20/A4 were lysed in a glass-teflon homogenizer using a homogenization buffer consisting of 50 mM Tris-HCl, 1 mM ethylenediamine-tetraacetic acid (EDTA), 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mg/mL of each of the protease inhibitors aprotinin, leupeptin and pepstatin (to prevent non-specific proteolytic degradation). The lysate was centrifuged at 100,000 g for 35 min to obtain a fraction enriched in plasma membranes. Protein assays were performed using a Bio-Rad kit.

C-20/A4 membranes were subjected to electrophoresis using pre-cast Novex NuPAGE™ Bis-Tris-HCl buffered (pH 6.4) gels in a Novex X-CELL II™ unit. Assayed protein samples were diluted in 4× sample buffer (1.17 M sucrose, 563 mM Tris Base, 423 mM Tris HCl, 278 mM SDS, 2.05 mM EDTA, 0.88 mM Serva Blue G250, 0.7 mM phenol red and ultrapure water). For the majority of experiments described 1.0 mm thick 4–12% gradient gels were used for optimum electrophoretic resolution. A MOPS (3-(N-morpholino) propane sulfonic acid) SDS (sodium dodecyl sulfate) running buffer was used. Electrophoretic transfer to nitrocellulose membranes (Hybond ECL, Amersham-Pharmacia, Bucks, UK) was performed in the same unit using a blot transfer module. The transfer buffer consisted of Bicine, Bis-Tris and EDTA.

C-20/A4 cells were allowed to attach to poly-l-lysine pre-coated slides (BDH, UK). Cells were fixed in ice-cold methanol for 10 min at −20 °C. After removal from the fixative the cells were washed once with PBS. Subsequently the cells were washed 3× with PBS. Non-specific binding was blocked by incubating the cells with PBS containing 10% normal goat serum (NGS; Sigma) for 1 h at room temperature. Primary α and β-isoform specific antibodies (see Table 2) were diluted in PBS containing 1% NGS and incubated with C-20/A4 cells for 2 h at room temperature. The cells were then washed 4× in PBS, and incubated with FITC-conjugated and TRITC-conjugated anti-mouse or anti-rabbit IgG (Sigma) in PBS containing 1% NGS (2 h at room temperature). The cells were finally washed 4× with PBS and mounted in mounting medium (Vectashield, Vector Laboratories Burlingame CA). In some immunocytochemical experiments, alkaline phosphatase- labeled anti-mouse and anti-rabbit secondary antibodies were used. For localization of alkaline phoshophatase activity Sigma Fast Red TR/Napthol AS-MX phosphate (4-chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphtoic acid 2,4-dimethylanilide phosphate) tablets were used.

Chondrocytes probed with α and β isoform specific antibodies using procedures described above were examined using a Zeiss fluorescence microscope fitted with an oil immersion objective (100×) and a MC 100 SPOT 35 mm microscope camera with automatic exposure control.

C-20/A4 chondrocytes grown to confluence as monolayer cultures were removed from culture flasks by treatment with trypsin EDTA (Life Technologies, UK), washed three times with PBS and fixed in 3.7% neutral buffered paraformaldehyde (Sigma, UK) in PBS for 10 min at 37 °C. The cells were washed three times with PBS and aliquots of 10⁻⁵ cells/100 mL PBS were placed into 1.5 mL eppendorf tubes, spun down (2000 rpm, 5 min) and the cell pellets were incubated with 100 mL aliquots of α subunit specific monoclonal antibodies (pan α, α6F, McB2 and McBX3; see Table 2 for dilutions used) at 4 °C overnight. Negative controls were incubated with 100 mL of a non-immune mouse IgG (DAKO, UK). Following three washes with PBS, the cells were incubated with 100 mL FITC-conjugated goat anti-mouse IgG diluted 1:100 (Sigma, UK) in PBS at 4 °C for 1 h in the dark. After three further washes in PBS, the cells were resuspended in 0.5 mL PBS and analyzed on a Becton-Dickinson FACScan. Plot profiles and histograms were generated using WinMDI flow cytometry software [31]. Statistical analysis was also performed using WinMDI.