Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

Roger Belusa, Oleg Aizman, Ronnie M. Andersson, and Anita Aperia

Department of Woman and Child Health, Karolinska Institutet, 171 77 Stockholm, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na+-K+-ATPase, that partial inhibition of Na+-K+-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na+-K+-ATPase, but not in cells expressing ouabain-insensitive Na+-K+-ATPase, whereas inhibition of Na+-K+-ATPase by lowering extracellular K+ concentration decreased attachment in all three cell types. Thirty percent inhibition of Na+-K+-ATPase significantly attenuated attachment. Na+-K+-ATPase inhibition caused a sustained increase in the intracellular Ca2+ concentration that obscured Ca2+ transients observed in untreated cells during attachment. Inhibitors of Ca2+ transporters significantly decreased attachment, but inhibition of Na+/H+ exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na+-K+-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca2+.

focal adhesion kinase; calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ENZYME sodium-potassium adenosine 5'-triphosphatase (Na+-K+-ATPase) is an integral membrane protein that in each mammalian cell establishes the electrochemical gradient across the plasma membrane by transporting 3 Na+ out of the cell and 2 K+ into the cell, utilizing ATP hydrolysis as an energy source. The activity of Na+-K+-ATPase also affects the intracellular Ca2+ concentration ([Ca2+]i) and pH by influencing the gradient for sodium-coupled transporters such as the Na+/Ca2+ and Na+/H+ exchangers. Hence, variations in the activity of Na+-K+-ATPase may have an effect on a number of important cell functions that are Ca2+ or pH dependent. Cell adhesion may be one such function. Several lines of evidence suggest that cell adhesion, required for development and maintenance of differentiated tissue, is Ca2+ dependent. Recent studies have indicated that long-term inhibition of Na+-K+-ATPase activity leads to detachment of cells (3, 7) and that early phases of cell attachment are perturbed in cells expressing Na+-K+-ATPase that lack the capacity to be regulated by phosphorylation.

The purpose of the present study has been to further elucidate the physiological significance of the relationship between Na+-K+-ATPase activity and cell attachment. We have examined the effects of inhibiting Na+-K+-ATPase activity, either with ouabain or by lowering the K+ concentration in the extracellular medium, on the early phase of attachment. The studies were performed on COS cells expressing three different types of Na+-K+-ATPase: COS Na+-K+-ATPase, rat Na+-K+-ATPase, and rat ouabain-resistant Na+-K+-ATPase. Recordings also were made of [Ca2+]i during cell attachment and after Na+-K+-ATPase inhibition with ouabain.

Our results indicate that a decrease in Na+-K+-ATPase activity leads to a concomitant decreased capacity in cell attachment to fibronectin and that this reduction in cell attachment can be at least partially mediated via changes in [Ca2+]i and a decrease in focal adhesion kinase (FAK) activation in these cells. The loss of attachment was not due to a decrease of integrin molecules on the cell surface, which suggests an effect on integrin activity. To our knowledge this is the first study showing a physiological relationship between short-term changes in Na+-K+-ATPase activity and cell attachment.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. Furosemide (5-[aminosulfonyl]-4-chloro-2-[(2-furanylmethyl)amino]benzoic acid), amiloride (N-amidino-3,5-diamino-6-chloropyrazinecarboxamide), W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride], nifedipine, fibronectin, KN-62 [1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine], ouabain [1beta ,3beta ,5beta ,11alpha ,14,19-hexahydroxycard-20(21)-enolide 3-(6-deoxy-alpha -L-mannopyranoside)], and biotin-X-X-NHS [6-(biotinamidocaproylamido)caproic acid N-hydroxysuccinimide ester] were purchased from Sigma (St. Louis, MO).

Ro-31-8210 (bisindolylmaleimide IX) and BIM (GF109203x, bisindolylmaleimide) were purchased from Calbiochem (San Diego, CA). FK-506 (Tacrolimus) was purchased from Fujisawa Pharmaceutical (Osaka, Japan). Okadaic acid was purchased from Scientific Marketing (Barnet, UK).

Cells. This study was performed on COS-7 cells, an embryonic monkey kidney cell line that is well suited for attachment studies because these cells do not require treatment with trypsin to detach from the plate. Three types of COS-7 cells were used: untransfected cells, cells stably transfected with wild-type (WT) rat Na+-K+-ATPase alpha 1-subunit, and cells transfected with rat Na+-K+-ATPase alpha 1-subunit in which leucine-799 was mutated to cysteine (L799C).

Transfection and selection. Transfection with WT and L799C rat Na+-K+-ATPase alpha 1-subunit was performed using the calcium phosphate method (16). Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Rockville, MD), supplemented with 10% fetal calf serum and 5% penicillin/streptomycin in 37°C humidified air with 5.0% CO2. COS cells express a highly ouabain-sensitive Na+-K+-ATPase, whereas the ouabain sensitivity of the transfected rat Na+-K+-ATPase alpha 1-subunit is ~500-fold less. Selection of transfected clones was performed as described previously (10, 27), based on this difference in ouabain sensitivity. Briefly, cells were grown in 10 µM ouabain for 3-4 wk, and the medium was changed every third day. Untransfected COS cells died within 2 days in 10 µM ouabain-containing medium, whereas cells expressing the relatively ouabain-insensitive rat Na+-K+-ATPase alpha 1-subunit survived. After this ouabain selection procedure, 200-300 single clones were pooled and replated in DMEM containing 10 µM ouabain. All experiments were performed on this mixture of clones.

Mutagenesis. Mutation of rat Na+-K+-ATPase alpha 1-subunit leucine 799 to cysteine was carried out by using a previously described protocol (3). Briefly, two DNA fragments were produced by PCR, one containing the mutation. PCR primers used were as follows: fragment 1 sense (5'-ATGATTGACCCTCCTCGAGCTGCT-3'); fragment 1 antisense (5'-AATAAATATCAAGAAGGGGGTGAT-3'); fragment 2 sense (5'-ATTGCAAACATTCCATGTCCCCTGG-3'); and fragment 2 antisense (5'-GGCCTGGATCATACCGATCTGT-3'). The fragments were ligated in a Pfu DNA ligase (Stratagene, San Diego, CA) reaction, and the combined fragment was substituted with the WT fragment using Eco47 and Bcl1 (New England Biolab, Beverly, MA) restriction enzymes. The construct was sequenced to eliminate the risk of unwanted errors.

Cell attachment. Measurement of cell attachment to fibronectin (5 µg/ml)-coated microtiter plates was performed as described earlier (3). Briefly, [3H]thymidine-labeled cells were incubated in serum-free DMEM or physiological saline solution (PSS) containing (in mM) 110 NaCl, 4 KCl, 1 CaCl2, 1.2 MgCl2, 1 Na2HPO4, 25 NaHCO3, 20 HEPES, and 10 glucose on a fibronectin-coated surface for 20-80 min, followed by washing with serum-free DMEM or PSS to remove unattached cells. Drugs were generally added just before incubation of cells on the coated surface. The Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaMKII) inhibitor KN-62 required a preincubation for 30 min (according to previous studies, see Refs. 4 and 5).

In experiments where we evaluated the effect of low extracellular K+ concentration, cells were incubated in PSS pH 7.4 at 37°C with different K+ concentrations (1-4 mM). Cell attachment was generally measured after 20 min. In one protocol, K+ concentration was returned to 5 mM and attachment was followed for an additional 60 min.

Cell growth and viability. To evaluate the effect of ouabain on cell viability, we measured cell growth and DNA fragmentation as an index of apoptosis. The untransfected COS-7 cells were incubated on plates for 3 h with vehicle or 10 µM ouabain. This incubation was followed by extensive washing with DMEM. Cells were subsequently returned to the ordinary growth medium (DMEM with 10% fetal calf serum and 5% penicillin/streptomycin in 37°C humidified air with 5.0% CO2) containing [3H]thymidine. Cell growth was measured as [3H]thymidine incorporation and increase in total protein concentration for five consecutive days. Light microscopy was used to study cell morphology.

Apoptosis in COS-7 cells treated with 10 µM ouabain for 3 h was evaluated by a modified DNA fragmentation assay by Duke (9). Briefly, untransfected COS-7 cell DNA was labeled with [3H]thymidine for 24 h. We incubated cells for 3 h with vehicle or 10 µM ouabain, and then the cells were lysed in a 25 mM sodium acetate buffer and centrifuged at 32,000 rpm for 20 min to separate intact DNA chromatin (pellet) from fragmented DNA (supernatant). Radioactivity was measured in both pellet and supernatant. A ratio of radioactivity between pellet and supernatant was calculated.

Rubidium uptake. All experiments were performed on cells in phosphate-buffered saline (PBS), which contained (in mM) 100 NaCl, 4.0 KCl, 1.0 CaCl2, 1.2 MgCl2, 20.0 HEPES, 25 NaHCO3, 1.0 NaH2PO4, and 10 D-glucose, and the pH was adjusted to 7.4 at 37°C. After the culture medium was removed, cells were washed twice and preincubated for 10 min in PBS to adjust the cells after the washing. Ouabain was added after 5 min of preincubation to obtain a final concentration of 50 or 100 nM. 86Rb+ flux measurements were initiated by adding 0.1 ml of PBS containing 10 µCi/ml 86Rb+ to each well. 86Rb+ uptake was linear for 5 min (data not shown), and recordings were made after 2 min. Experiments were performed at 37°C. Reactions were stopped by rinsing cells three times with ice-cold PBS containing 5 mM BaCl2. Cells were then lysed by the addition of 0.6 ml of 1 M NaOH. Radioactivity in cell lysates was measured with a scintillation counter (LKB; Wallac, Turku, Finland) and calculated as counts per minute (cpm) per milligram of protein. Protein was determined by using a Bio-Rad assay (Hercules, CA) with bovine serum albumin as standard.

Cell surface biotinylation and immunoprecipitation. To examine the possibility of integrin internalization, we detached cells and incubated them in a suspension with 1 mM ouabain at 37°C for 20 min. The cells were transferred to ice, and biotin-X-X-NHS (0.5 mg/ml final concentration; Sigma) was added. After 20 min of incubation with biotin, cells were washed several times with 0.1 M glycine-containing ice-cold PBS pH 7.4 and then lysed on ice for 20 min with a 1% Nonidet P-40 (NP-40)-PBS buffer containing a mix of protease inhibitors (Complete; Roche Diagnostics, Mannheim, Germany). Cell lysates were centrifuged at 14,000 rpm at 4°C for 10 min. The supernatant was saved and incubated for 60 min with Sepharose-protein A 6MB beads (Pharmacia Biotech, Uppsala, Sweden) at 4°C with shaking. The samples were again centrifuged as described above, and supernatants were saved. The supernatants were incubated with an anti-beta 1-integrin antibody (kind gift from Dr. Erkki Ruoslahti, Burnham Institute, La Jolla, CA) at 4°C for 18 h. Precipitation of the serum was carried out with Sepharose-protein A bead incubation at 4°C for 60 min. Beads were washed three times with ice-cold 1% NP-40-containing PBS buffer. Before the incubation with anti-beta 1-integrin antibody, aliquots were removed and used for protein concentration measurements with the Bio-Rad DC protein assay kit.

We evaluated the possibility of a direct interaction between Na+-K+-ATPase and beta 1-integrin in the cells by using a coimmunoprecipitation method. An anti-beta 1-integrin antibody was used in immunoprecipitation, and anti-Na+-K+-ATPase alpha 1-subunit antibody H6 (kind gift from Dr. Kaplan, Dept. of Biochemistry and Molecular Biology, Oregon Health Science University, Portland, OR) was used for immunoblotting. Several different concentrations (0.1-1.0%) of the detergent NP-40 were used to vary the stringency of the immunoprecipitation.

Samples were separated on a 6% polyacrylamide gel and transferred to a Hybond-P filter (Amersham Pharmacia Biotech, Amersham, UK), and the level of biotinylation was detected using peroxidase-conjugated ExtrAvidin (Sigma). A radiographic film (Super RX Medical X-Ray Film, FujiFilm, Tokyo, Japan) was exposed to the filter. The signal was quantified using Quantity One 4.1.1 software (Bio-Rad) and the Fluor-S MultiImager (Bio-Rad).

FAK phosphorylation. Cells were detached, resuspended in PSS, and replated on a fibronectin (5 µg/ml)-coated surface. After 20 min, a 5× concentrated lysis buffer was added to the cells to obtain a final lysis buffer concentration: 1× PBS (pH 7.4), 1% NP-40, protease inhibitor cocktail (Complete), and phosphatase inhibitors (25 mM NaF, 1 mM Na3VO4, and 10-7 M okadaic acid). The plates were transferred to ice for 20 min. Cell lysates were centrifuged at 14,000 rpm at 4°C for 10 min, and supernatant was saved and incubated for 180 min with Sepharose-protein A beads (Pharmacia-Biotech) at 4°C with shaking. Samples were again centrifuged as described above, and the supernatants were saved. Supernatants were incubated with an anti-FAK antibody (Upstate Biotechnology, Lake Placid, NY) at 4°C for 18 h. Precipitation of the serum was carried out with Sepharose-protein A bead incubation at 4°C for 180 min. Beads were washed three times with ice-cold lysis buffer. The samples were separated on 6% polyacrylamide gels. The gels were transferred to a Hybond-P filter (Amersham Pharmacia Biotech). The total amount of FAK was detected with the anti-FAK antibody, and the level of phosphorylated FAK was detected with a tyrosine-397-phosphospecific anti-FAK serum (BioSource International, Camarillo, CA). The secondary antibody was peroxidase-conjugated anti-rabbit antibody (Sigma). A radiographic film (Super RX Medical X-Ray Film; FujiFilm) was exposed to the filter. The signal was quantified by using the same technique as described in Cell surface biotinylation and immunoprecipitation.

Measurement of [Ca2+]i. Cells were grown on a 40-mm coverslip for 2-3 days and then incubated in DMEM culture medium with fura 2-AM (2 µM) and pluronic acid (0.2% wt/vol) for 1 h at 37°C in the incubator. After the fura 2-AM was loaded, the cells were washed in PSS containing (in mM) 110 NaCl, 4 KCl, 1 CaCl2, 1.2 MgCl2, 1 Na2HPO4, 25 NaHCO3, 20 HEPES, and 10 glucose, pH 7.4, at 37°C for 15 min. Ratiometric imaging was performed by using a heated chamber (FCS2; Bioptechs) mounted on a Zeiss Axiovert 135 microscope with a 40/1.4 epifluorescence oil-immersion objective. The excitations were set at 340 and 380 nm, and the emission was at 510 nm. Emission fluorescence was collected via a GenIISys image intensifier system connected to a charge-coupled device camera (MTI CCD72; Dage-MTI) and acquisition software from Inovision. Fluorescence was recorded as the 340-to-380-nm ratio every 60 s. For a more complete description of the methods, see Refs. 11 and 26.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of ouabain on attachment was first tested by using untransfected COS cells that express a Na+-K+-ATPase that is highly sensitive to ouabain. We found that ouabain at 10 µM, a concentration that gives full inhibition of endogenous COS Na+-K+- ATPase, caused a dramatic (93 ± 3%, mean ± SE, n = 5) decrease in the number of attached cells 20 min after plating. We next tested the effect of partial inhibition of Na+-K+-ATPase. Activity of Na+-K+-ATPase was measured as ouabain-sensitive uptake of trace amounts of 86Rb+. Ouabain at 50 and 100 nM caused 25.7 ± 3.8% and 57.4 ± 8.2% inhibition of Na+-K+-ATPase activity and decreased the number of attached cells by 29.3 ± 5.7% and 46.4 ± 2.2%, respectively (Fig. 1).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Ouabain inhibition of Rb+ flux (A) and cell attachment to fibronectin (B) in untransfected COS cells. Data are means ± SE from 3 independent experiments and are shown as %ouabain-sensitive Rb+ uptake and %cell attachment compared with untreated control cells.

To evaluate to which extent Na+-K+-ATPase inhibition affected cell viability, we studied cell survival after treatment with ouabain. Untransfected COS-7 cells were incubated 3 h with 10 µM ouabain, washed, and incubated with medium without ouabain for 1-5 days. Protein content and levels of [3H]thymidine incorporation were similar in untreated and treated cells during the 5 days following this procedure. No increase was found in the level of DNA fragmentation (a sign of apoptosis) in cells treated with ouabain for 3 h (data not shown).

COS cells transfected with rat Na+-K+-ATPase alpha 1-subunit were studied in the following protocols. All studies were performed in the presence of 10 µM ouabain to inhibit the activity of endogenous COS Na+-K+-ATPase. Rat Na+-K+-ATPase is not significantly inhibited by 10 µM ouabain (94 ± 3% activity compared with untreated cells, mean ± SE, n = 3). The COS cells were transfected with either WT rat Na+-K+-ATPase alpha 1-subunit or L799C rat Na+-K+-ATPase alpha 1-subunit. This mutant is virtually completely resistant to ouabain (Andersson et al., unpublished observations).

Ouabain was found to produce a dose-dependent inhibition of attachment in cells expressing WT rat Na+-K+-ATPase (Fig. 2A). Inhibition of attachment was significant already at 50 µM, a concentration of ouabain that produces an ~30% inhibition of Na+-K+-ATPase activity. At 40 min of incubation on the fibronectin-coated surface, 1 mM ouabain inhibited attachment in cells expressing WT rat Na+-K+-ATPase to the same extent as in cells incubated for 20 min (27.1 ± 2.2% attachment compared with untreated cells, mean ± SE, n = 5). Ouabain at 1 mM had no effect on the attachment of cells expressing the ouabain-insensitive L799C rat Na+-K+-ATPase alpha 1-subunit (Fig. 2B).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   A: ouabain inhibition of cell attachment to fibronectin of COS cells expressing wild-type (WT) rat Na+-K+-ATPase alpha 1-subunit. A dose-dependent decrease in cell attachment is observed. Each value represents the mean ± SE of 3-5 independent experiments, and the results are presented as %attachment compared with untreated control cells. B: comparison of the effect of 1 mM ouabain on cell attachment to fibronectin in COS cells expressing WT rat Na+-K+-ATPase alpha 1-subunit and COS cells expressing ouabain-resistant L799C rat Na+-K+-ATPase alpha 1-subunit. Each value represents the mean ± SE of 5 independent experiments, and the results are presented as %attachment compared with untreated control cells.

Extracellular K+ is one rate-limiting factor for Na+-K+-ATPase activity. We investigated the effect of decreasing concentrations of extracellular K+ on cell attachment to fibronectin. Lowering the extracellular K+ concentration also led to a dose-dependent decrease of cell attachment (Fig. 3A). Addition of extracellular K+ to the incubation medium resulted in a reactivation of Na+-K+-ATPase and recovery of cell attachment (Fig. 3B). COS-7 cells expressing ouabain-resistant L799C Na+-K+-ATPase also showed a dose-dependent decrease in cell attachment to fibronectin. The effect was in the same range as for cells expressing WT rat Na+-K+-ATPase (data not shown). From these studies we conclude that the effects of ouabain on attachment are secondary to the inhibition of Na+-K+-ATPase activity and cannot be attributed to the conformational change of Na+-K+-ATPase that occurs after ouabain binding.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   A: attachment of COS cells expressing WT rat Na+-K+-ATPase to fibronectin in the presence of different concentrations of extracellular K+. A dose-dependent decrease in cell attachment is noted. Values are presented as %attachment of total and represent means ± SE of 3 independent experiments. B: attachment of COS cells expressing WT rat Na+-K+-ATPase to fibronectin. The extracellular K+ concentration was initially 1 mM and was then changed to 5 mM (see horizontal bar). Values are presented as %attachment of total and represent means ± SE of 3 independent experiments.

We next examined whether inhibition of Na+ influx had any effect on attachment as ouabain inhibition of Na+-K+-ATPase. Amiloride at 10 µM, a concentration known to inhibit both the Na+/H+ exchanger and the epithelial Na+ channel, had no effect on cell attachment to fibronectin (103 ± 8.4% compared with control, mean ± SE, n = 5). Furosemide, used as an inhibitor of the Na+-K+-Cl- cotransporter, had a stimulating effect on attachment (149 ± 9.8% compared with control, mean ± SE, n = 3) at low concentrations (5 µM). At higher concentrations (100 µM), there was no effect on attachment (119 ± 15.5% compared with control, mean ± SE, n = 6).

Intracellular Ca2+ is known to have an effect on cell attachment (14, 17, 23, 24). Recordings of [Ca2+]i during the process of attachment revealed that in untreated COS-7 cells expressing WT rat Na+-K+- ATPase alpha 1-subunit, there was a transient (~15 min) increase in [Ca2+]i (Fig. 4A). In contrast, all cells that had been exposed to ouabain showed a sustained increase in [Ca2+]i (Fig. 4B). Ouabain at 1 mM increased [Ca2+]i in cells expressing WT rat Na+-K+-ATPase alpha 1-subunit but had no effect on [Ca2+]i in cells expressing resistant L799C rat Na+-K+-ATPase alpha 1-subunit (Fig. 5).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Representative single-cell intracellular Ca2+ concentration ([Ca2+]i) tracings during cell adhesion to fibronectin. Studies were performed in COS cells expressing WT rat Na+-K+-ATPase alpha 1-subunit. Fura 2-AM-loaded cells were plated on glass coverslips coated with fibronectin. The measurement of [Ca2+]i in arbitrary units (au) was initiated 2-5 min after plating. Each experiment was performed at least 4 times; a total of 120 control cells (A) and 98 ouabain-treated cells (B) were examined.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of 1 mM ouabain treatment on [Ca2+]i in cells expressing WT rat Na+-K+-ATPase alpha 1-subunit or L799C rat Na+-K+- ATPase alpha 1-subunit. Data are averages from 3 independent experiments. An increase in [Ca2+]i was consistently seen in cells expressing WT Na+-K+-ATPase subunit (n = 57 cells), but no change in [Ca2+]i was detected in cells expressing ouabain-resistant rat L799C Na+-K+-ATPase (n = 30 cells).

We next evaluated the role of Ca2+ signaling pathways on the ouabain-induced decrease in attachment by using inhibitors to some Ca2+-dependent signaling molecules. KN-62, an inhibitor of Ca2+/CaMKII, had by itself an inhibitory effect on cell attachment (62 ± 5.6% compared with control, mean ± SE, n = 3). KN-62 did not have any additional effect on the ouabain-induced decrease in cell attachment (70.9 ± 7.2% compared with control, mean ± SE, n = 3). To evaluate the role of the Ca2+-dependent protein phosphatase calcineurin, we used FK-506, a potent inhibitor of calcineurin. Cell attachment to fibronectin was decreased by 100 nM FK-506 (57 ± 5.4% compared with control, mean ± SE, n = 4). FK-506 did not have any additional effect on ouabain-induced decrease in attachment (74.6 ± 5.6% compared with control, mean ± SE, n = 3). W-7 (1 µM), a calmodulin inhibitor, had a dramatic inhibitory effect on cell attachment to fibronectin in COS-7 cells (12.5 ± 1.4% attachment compared with untreated cells, mean ± SE, n = 4). Because W-7 alone caused almost complete inhibition of attachment, we did not evaluate the effect of W-7 on ouabain-induced decrease in cell attachment.

Nifedipine (50 µM), an inhibitor of L-type voltage-gated Ca2+ channels, caused a small inhibitory effect on cell attachment (77.6 ± 7.8% attachment compared with untreated cells, mean ± SE, n = 3). Nifedipine had no additional effect on the ouabain-induced decrease in cell attachment (56.4 ± 5.1% compared with untreated cells, mean ± SE, n = 3).

The inositol 1,4,5-trisphosphate (IP3) receptor regulates the release of intracellular Ca2+ storage of the endoplasmic reticulum. A cell-permeable inhibitor of IP3 receptors, 2-aminoethyoxydiphenyl borate (2-APB) (15), had, when added singly at the 50 µM dose, an inhibitory effect on cell attachment to fibronectin. 2-APB also had an additional effect on the ouabain-induced decrease in cell attachment (Fig. 6).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of the inositol 1,4,5-trisphosphate (IP3) receptor inhibitor 2-aminoethyoxydiphenyl borate (2-APB; 50 µM) on attachment of COS cells expressing WT rat Na+-K+-ATPase and ouabain-induced decrease in cell attachment to fibronectin. Experiments were performed in the presence (+) or absence (-) of 50 µM ouabain. Each bar represents the mean ± SE of 3 independent experiments, and the results are presented as %attachment compared with untreated control cells.

Protein kinase C (PKC) has been shown to regulate alpha 5beta 1-integrin activity in Chinese hamster ovary cells (28). We examined the effects of two PKC inhibitors, Ro-31-8210 (100 nM) and BIM (1 µM), that inhibit Ca2+-dependent PKC isoforms. Both drugs resulted in a moderate inhibition of cell attachment but had no additional effect on the ouabain-induced decrease in cell attachment (data not shown).

FAK is one of the key signal transduction mediators during beta 1-integrin-mediated cell adhesion to fibronectin. We used an antibody specific to phosphorylated tyrosine-397 FAK, a site that is autophosphorylated when the enzyme is activated. As a control, an antibody recognizing both phosphorylated and nonphosphorylated FAK was used. COS-7 cells expressing rat Na+-K+-ATPase were incubated on a fibronectin-coated surface for 20 min, with or without 1 mM ouabain pretreatment, and were then prepared for Western blot. The ratio between the signals from the total FAK in cells lysates from control and cells that had been incubated with ouabain (1 mM) or low (1 mM) extracellular K+ was not significantly different from 1 (data not shown). The ratio between the signals from the phospho-specific FAK antibody in lysates from control and ouabain-treated cells was 0.46 ± 0.1 (mean ± SE, n = 3) in control cells and was 0.71 ± 0.06 (mean ± SE, n = 4) in cells exposed to low (1 mM) extracellular K+ solution. This finding indicates that exposure to both ouabain and low extracellular K+ causes a significant decrease in FAK phosphorylation (Fig. 7). We next investigated whether the decreased attachment to fibronectin was due to internalization of beta 1-integrin on the cell surface. COS-7 cells have high levels of alpha 5- and beta 1-integrins on their cell surface (data not shown). The alpha 5beta 1-integrin is one major fibronectin receptor. Ouabain- and vehicle-treated cells were labeled by biotinylation after 20 min of pretreatment. No effect of ouabain on cell surface labeling of beta 1-integrin was seen (control 211 ± 20.7 vs. treated 214 ± 21.7 optical density units, means ± SE, n = 3).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of ouabain and low extracellular K+ concentration (KClex) on focal adhesion kinase (FAK) autophosphorylation during attachment to fibronectin. Representative Western blots (top) and densitometric analysis (bottom) of 3-4 independent experiments show a decrease in FAK autophosphorylation in cells exposed to 1 mM ouabain or low (1 mM) extracellular K+. FAK-p, phosphorylated FAK.

To evaluate the possibility of a direct interaction between Na+-K+-ATPase and integrin, we performed coimmunoprecipitation experiments using both the anti-beta 1-integrin antibody and 6H, the anti-alpha 1-subunit Na+-K+-ATPase antibody. We found no evidence for a coprecipitation of Na+-K+-ATPase and beta 1-integrin (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that a decrease in Na+-K+-ATPase activity leads to a concomitant decrease in COS-7 cell attachment to fibronectin (Figs. 1 and 2). This was shown by two independent experiments, first by inhibiting Na+-K+-ATPase activity by ouabain, a specific ligand of Na+-K+-ATPase, and second by lowering extracellular K+, a substrate for Na+-K+- ATPase. In both cases, a dose-dependent decrease in cell attachment to fibronectin was noted. Notably, cell attachment was significantly influenced already at 30% inhibition of Na+-K+-ATPase activity (Figs. 1 and 2A). No effect on cell attachment by ouabain was seen in COS-7 cells expressing the ouabain-insensitive L799C Na+-K+-ATPase (Fig. 2B).

Inhibition of Na+-K+-ATPase activity will directly affect [Na+]i and [K+]i concentrations and indirectly affect [Ca2+]i and intracellular pH (pHi). It is possible that more than one of these factors might have contributed to the effect of Na+-K+-ATPase inhibition on cell attachment, but so far it is only well established that intracellular Ca2+ has an effect on cell attachment. One of the earliest responses to integrin-mediated cell spreading is a transient increase in [Ca2+]i (8, 20, 21, 24). Such a response also was observed in the present study in untreated cells (Fig. 4A), but not in the ouabain-exposed cells (Fig. 4B).

Na+-K+-ATPase inhibition is generally associated with an increase in [Ca2+]i that may be of different types, depending on cell type. In most cells the effect of Na+-K+-ATPase inhibition has been attributed to an increase in intracellular Na+ concentration, which in turn dissipates the gradient for Na+/Ca2+ exchanger. This may lead to a sustained increase in [Ca2+]i, as observed in this study in ouabain-treated COS-7 cells (Figs. 4 and 5). Physiological Ca2+ signaling generally occurs as Ca2+ transients, as observed in the untreated COS-7 cells during attachment, or as Ca2+ waves, and is dependent on the activation of other transporters, such as the voltage-gated Ca2+ channels in the plasma membrane and the IP3 or ryanodine receptor in the membrane of the endoplasmic reticulum. We hypothesize that the sustained increase in [Ca2+]i, observed in the ouabain-exposed cells, will override and abrogate the physiological Ca2+ signaling pathways. Inhibitors of the voltage-gated L-type Ca2+ channel and the IP3 receptor did, when added singly, decrease attachment to a similar extent as ouabain. These findings support the hypothesis that the physiological Ca2+ signaling pathways that are involved in the process of cell attachment are perturbed or abolished by the Ca2+ effects induced by ouabain. The calmodulin inhibitor W-7 caused, when added alone, an ~90% decrease in attachment. Ca2+/CaMKII has been reported to regulate alpha 5beta 1-integrin signaling and cell adhesion to fibronectin (4, 5). KN-62, a Ca2+/CaMKII inhibitor, when added singly, had a small inhibitory effect on attachment but did not influence the ouabain-induced effect.

There are a few studies suggesting that changes in pHi are involved in regulating the cellular adhesion process (13, 22). Inhibition of the Na+/H+ exchanger with amiloride, however, did not result in any effect on cell attachment in this study.

One early event in integrin-mediated cell adhesion to fibronectin is the activation of FAK (6, 12, 18) and autophosphorylation of FAK at tyrosine-397 (19). There are some indications that FAK phosphorylation can be affected by intracellular Ca2+ signaling pathways (1). We have shown that FAK phosphorylation was decreased after treatment with ouabain (Fig. 7) but that the level of cell surface expression of beta 1-integrin was unchanged. This finding suggests that Na+-K+-ATPase inhibition influences the activation of fibronectin-binding integrins on the cell surface and/or that the signal between the integrin and FAK was perturbed.

In summary, this study has demonstrated a close link between the activity of Na+-K+-ATPase and cell attachment to fibronectin that might, at least partially, be mediated via changes in [Ca2+]i. The activity of Na+-K+-ATPase can be modulated by a number of physiological and pharmacological factors, including activation of G protein-coupled receptors, endogenous ouabain, and cytoskeleton proteins (2, 25). It is now important to find out what effect these factors have on cell attachment.


    ACKNOWLEDGEMENTS

This study was supported by grants from the Swedish Medical Research Council, the Märta and Gunnar V. Philipson Foundation, Stiftelsen Frimurare Barnhuset Stockholm, and the Stiftelsen Axel Tielmans Minnesfond.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Aperia, Dept. of Woman and Child Health, Astrid Lindgren Children's Hospital, Q2:09, 171 76 Stockholm, Sweden (E-mail: anita.aperia{at}ks.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00117.2001

Received 9 March 2001; accepted in final form 21 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anfosso, F, Bardin N, Vivier E, Sabatier F, Sampol J, and Dignat-George F. Outside-in signaling pathway linked to CD146 engagement in human endothelial cells. J Biol Chem 276: 1564-1569, 2001[Abstract/Free Full Text].

2.   Aperia, A. Regulation of sodium/potassium ATPase activity: impact on salt balance and vascular contractility. Curr Hypertens Rep 3: 165-171, 2001[Medline].

3.   Belusa, R, Wang ZM, Matsubara T, Sahlgren B, Dulubova I, Nairn AC, Ruoslahti E, Greengard P, and Aperia A. Mutation of protein kinase C phosphorylation site on rat alpha 1 Na+,K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness. J Biol Chem 272: 20179-20184, 1997[Abstract/Free Full Text].

4.   Bouvard, D. Calcium/calmodulin-dependent protein kinase II controls integrin alpha 5beta 1-mediated cell adhesion through the integrin cytoplasmic domain associated protein-1alpha . Biochem Biophys Res Commun 252: 46-50, 1998[ISI][Medline].

5.   Bouvard, D, and Block MR. Calcium/calmodulin-dependent protein kinase II controls alpha 5beta 1 integrin-mediated inside-out signaling. J Cell Sci 111: 657-665, 1998[Abstract/Free Full Text].

6.   Burridge, K, and Romer LH. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 119: 893-903, 1992[Abstract].

7.   Contreras, RG, Flores-Maldonado C, Lazaro A, and Cereijido M. Relationship between Na+,K+-ATPase and cell attachment. J Cell Sci 112: 4223-4232, 1999[Abstract/Free Full Text].

8.   Coppolino, MG, Demaurex N, Grinstein S, St-Arnaud R, and Dedhar S. Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 386: 843-847, 1997[ISI][Medline].

9.   Duke, RC, and Cohen JJ. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci USA 80: 6361-6365, 1983[Abstract].

10.   Fisone, G, Cheng SX, Nairn AC, Czernik AJ, Hemmings HC, Hoog JO, Bertorello AM, Kaiser R, Bergman T, and Jornvall H. Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K+-ATPase and effects of site-directed mutagenesis. J Biol Chem 269: 9368-9373, 1994[Abstract/Free Full Text].

11.   Grynkiewicz, G, Poenie M, and Tsien R. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

12.   Kornberg, L, Parsons JT, Schaller M, and Juliano RL. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem 267: 23439-23442, 1992[Abstract/Free Full Text].

13.   Margolis, LB, and Cragoe E. Intracellular pH and cell adhesion to solid substrate. FEBS Lett 234: 449-450, 1988[ISI][Medline].

14.   Marie, C, Soyez S, and Block MR. Semi-intact CHO and endothelial cells: a tool to probe the control of integrin activity? Exp Cell Res 192: 173-181, 1991[ISI][Medline].

15.   Maruyama, T, Kanaji T, Nakade S, Kanno T, and Mikoshiba K. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem (Tokyo) 122: 498-505, 1997[Abstract].

16.   Okayama, H, and Chen C. Gene Transfer and Expression Protocols. Clifton, NJ: Humana, 1991, p. 15-21.

17.   Rowin, ME, Yednock T, and Bohnsack JF. Intracellular calcium requirements for beta 1 integrin activation. J Cell Physiol 175: 193-202, 1998[ISI][Medline].

18.   Schaller, MD, Cobb BS, Vines RR, Reynolds AB, and Parsons JT. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci USA 89: 5192-5196, 1992[Abstract].

19.   Schaller, MD, Shannon JD, Fox JW, Vines RR, and Parsons JT. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 14: 1680-1688, 1994[Abstract].

20.   Schwartz, MA. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Cell Biol 120: 1003-1010, 1993[Abstract].

21.   Schwartz, MA, and Fazeli B. A 50-kDa integrin-associated protein is required for integrin-regulated calcium entry in endothelial cells. J Biol Chem 268: 19931-19934, 1993[Abstract/Free Full Text].

22.   Schwartz, MA, and Ingber DE. Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha 5beta 1, independent of cell shape. Proc Natl Acad Sci USA 88: 7849-7853, 1991[Abstract].

23.   Sjaastad, MD. Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium. Bioessays 19: 47-55, 1997[ISI][Medline].

24.   Sjaastad, MD, and Nelson WJ. Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx. Mol Biol Cell 7: 1025-1041, 1996[Abstract].

25.   Therien, AG, and Blostein R. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279: C541-C566, 2000[Abstract/Free Full Text].

26.   Uhlen, P, Laestadius A, Jahnukainen T, Soderblom T, Backhed F, Celsi G, Brismar H, Normark S, Aperia A, and Richter-Dahlfors A. alpha -Haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells. Nature 405: 694-697, 2000[ISI][Medline].

27.   Vilsen, B. Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha -subunit of the rat kidney Na+,K+-ATPase. FEBS Lett 314: 301-307, 1992[ISI][Medline].

28.   Vuori, K, and Ruoslahti E. Activation of protein kinase C precedes alpha 5beta 1 integrin-mediated cell spreading on fibronectin. J Biol Chem 268: 21459-21462, 1993[Abstract/Free Full Text].


Am J Physiol Cell Physiol 282(2):C302-C309
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (8)
Google Scholar
Articles by Belusa, R.
Articles by Aperia, A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Belusa, R.
Articles by Aperia, A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online