Na,K-ATPase is a well studied molecule, best appreciated for its role in the regulation of ion homeostasis in mammalian cells. In this study, we demonstrate that Na,K-ATPase is localized to the actin-rich lamellipodia and is involved in controlling cell motility in carcinoma cells. Our results are consistent with a motility suppressor role for Na,K-β in epithelial cells. The cytoplasmic tail of Na,K-β is necessary for the lamellipodial localization of Na,K-ATPase, enhanced Rac1 activity, and suppression of cell motility in MSV-MDCK cells. We also demonstrate that suppression of cell motility is dependent on 1) the activation of PI3-kinase mediated by the association of N-terminus of Na,K-α with p85, and 2) the binding of the cytoplasmic tail of Na,K-β to annexin II. Finally, we show that inhibition of cell motility by Na,K-ATPase is independent of its ion transport function. Our results demonstrate that Na,K-β suppresses cell motility by a novel signaling mechanism involving cross-talk between the two subunits of Na,K-ATPase with proteins associated with PI3-kinase signaling.
Several lines of evidence strongly support that Na,K-β is specifically involved in the suppression of cell motility in MSV-MDCK cells. 1) MSV-MDCK cells have very low levels of Na,K-β and are highly motile. We have shown in three independent clones (MSV-β cl1 [Rajasekaran et al., 2001b
], MSV-β cl2 [Rajasekaran et al., 2001b
, this study], and MSV-β-GFP [this study]) that repletion of Na,K-β in MSV-MDCK cells suppresses motility. 2) Na,K-βΔCD-GFP, a cytoplasmic tail deletion mutant of Na,K-β, neither showed lamellipodial localization nor reduced the motility of MSV-MDCK cells. 3) The levels of active Rac1 in MSV-βΔCD-GFP cells were comparable with that of parental MSV-MDCK cells, indicating that the cytoplasmic tail of Na,K-β is essential for the signaling leading to the activation of Rac1. 4) Increased levels of Na,K-α in MSV-β cells are involved in the activation of PI3-kinase. Because Na,K-β facilitates the translation efficiency of Na,K-α in the endoplasmic reticulum (Rajasekaran et al., 2004
), Na,K-β function also is involved indirectly in the activation of PI3-kinase by Na,K-α in MSV-β cells. 5) By increasing the levels of Na,K-α in MSV-βΔCD-GFP cells, which have very low levels of Na,K-α, we provided evidence that activation of PI3-kinase alone is not sufficient and that the association of Na,K-β with annexin II is also involved in the suppression of cell motility in MSV-MDCK cells. Together, these results demonstrate that reduced motility is primarily mediated by Na,K-β in MSV-MDCK cells.
We have recently shown that the transcription factor Snail known to suppress E-cadherin expression in carcinoma also reduces the transcription of Na,K-β (Espineda et al., 2004
). Snail expression during normal development as well as during epithelial to mesenchymal transition induces motility of epithelial cells by reducing the expression of proteins such as E-cadherin (Nieto, 2002
). Reduction of Na,K-β transcription by Snail (Espineda et al., 2004
) and suppression of motility by repletion of Na,K-β in MSV-MDCK cells (this study), strongly suggests that high Na,K-β expression is associated with reduced motility in well differentiated epithelial cells. Based on these results, we suggest that Na,K-β is a motility suppressor in normal epithelial cells and that reduced levels of this protein are associated with increased motility of carcinoma cells.
Repletion of Na,K-β expression in MSV-MDCK cells induced dramatic reorganization of the actin cytoskeleton. Reduced motility of MSV-β cells correlated with abundant lamellipodia and substantially decreased amount of stress fibers. In general, induction of lamellipodia is associated with enhanced cell migration (Liliental et al., 2000
; Sastry et al., 2002
; Ridley et al., 2003
). However, lamellipodia also can be coupled with decreased cell motility. For example, Chisel protein (normally expressed in heart and skeletal muscles), when exogenously expressed in myoblasts, induced lamellipodia, localized to lamellipodia and suppressed motility (Palmer et al., 2001
). The mechanism by which Chisel protein suppresses motility is not known. Cell migration requires the polarization of the cells into a leading edge marked by lamellipodia and a trailing edge composed of focal adhesions (Ridley et al., 2003
). In the absence of such a polarization, cell spreading is enhanced, leading to suppression of cell motility (Sander et al., 1999
). We observed that MSV-β cells were more spread out compared with MSV-GFP and MSV-βΔCD-GFP cells (), which have reduced lamellipodia. This finding is consistent with the notion that exogenous expression of Na,K-β restricts cell movement by increased cell spreading and attachment to the substratum. Experiments are in progress in our laboratory to unravel the mechanism by which induction of lamellipodia leads to suppression of motility in MSV-β cells.
MSV-β cells were less motile and had highly reduced stress fibers. MSV-MDCK, MSV-βΔCD-GFP, and LY294002-treated MSV-β cells had abundant stress fibers, which correlated with increased motility of these cells. Increased amount of stress fibers provides enhanced contractility and promotes the migratory behavior of cells (Zhong et al., 1997
). Therefore, it is possible that increased stress fibers might contribute to increased motility observed in MSV-MDCK, MSV-βΔCD-GFP, and LY294002-treated MSV-β cells. Formation of stress fibers is known to be regulated by the activity of RhoA (Ridley and Hall, 1992
). However, we did not observe a difference in the levels of active RhoA in these cell lines. Because RhoA-independent mechanisms are known to modulate stress fiber formation and motility in mammalian cells (Ory et al., 2002
), the possibility that such mechanisms are involved in the formation of stress fibers and enhanced motility in MSV-MDCK cells cannot be ruled out.
Na,K-β–mediated suppression of cell motility in MSV-MDCK cells involved PI3-kinase–dependent activation of Rac1. Strikingly, it has been shown that E-cadherin–mediated cell-cell adhesion also leads to PI3-kinase dependent Rac1 activation and suppression of cell motility (Braga et al., 1997
; Hordijk et al., 1997
; Takaishi et al., 1997
; Sander et al., 1999
; Yap and Kovacs, 2003
). However, MSV-β cells, like MSV-MDCK cells, lack E-cadherin and cadherin-mediated cell-cell adhesion and adherens junction formation (Rajasekaran et al., 2001b
). Therefore, Rac1 activation in MSV-β cells is independent of cadherin-mediated adhesion and is dependent on the expression of Na,K-β in these cells. Although MSV-β cells lack E-cadherin, interestingly, they do aggregate in an in vitro cell aggregation assay, indicating that Na,K-β might have cell-cell adhesion function (Rajasekaran et al., 2001b
). Whether the activation of Rac1 by exogenous expression of Na,K-β is a result of Na,K-β–mediated cell-cell adhesion remains to be tested.
Our results suggest that increased p85 binding to Na,K-α due to increased expression of Na,K-α in MSV-β cells is involved in the activation of p85. Although MSV-βΔCD-GFP and MSV-MDCK cells showed comparable levels of tyrosine-phosphorylated p85, exogenous expression of Na,K-α in MSV-βΔCD-GFP cells increased the tyrosine phosphorylation of p85, supporting the idea that elevated levels of Na,K-α are involved in the increased phosphorylation of p85. The mechanism by which Na,K-α increases the tyrosine phosphorylation of p85 is currently not known. It is possible that specific kinases activated in MSV-β cells might be involved in increasing the tyrosine phosphorylation of p85 and facilitating the association of Na,K-α with p85. Future studies are necessary to understand this process. Interestingly, it has been reported that dopamine-mediated endocytosis and inhibition of Na,K-ATPase involves activation of PI3-kinase by Na,K-α binding to p85 (Yudowski et al., 2000
), indicating that activation of PI3-kinase by binding to p85 is involved in the clearance of Na,K-ATPase from the plasma membrane. However, our results indicate that association of p85 with Na,K-α occurs under conditions where the pump is functional and is involved in the formation of lamellipodia. Although PI3-kinase activation is necessary, it is not sufficient for the suppression of motility. In MSV-βΔCD-GFP cells, exogenous expression of Na,K-α increased tyrosine-phosphorylated p85 levels, however, did not suppress motility, indicating that the downstream events involving the cytoplasmic tail of Na,K-β are essential for the suppression of motility in MSV-MDCK cells.
One of the striking findings reported in this study is the association of annexin II with the cytoplasmic tail of Na,K-β. Annexin II binds to PIP3
), which is produced in cells due to the activation of PI3-kinase (Welch et al., 2003
). Annexin II is found in the preparations of lamellae (Ghitescu et al., 2001
) and is involved in the regulation of actin polymerization necessary for the delivery of macropinosomes to the plasma membrane (Merrifield et al., 2001
). Annexin II also has been shown to inhibit cell migration (Balch and Dedman, 1997
; Liu et al., 2003
). The carboxy terminus of annexin II binds F-actin (Filipenko and Waisman, 2001
), and this association might stabilize annexin II at the actin-rich lamellipodia. The binding of annexin II to CD44/H-CAM results in Rac1 dependent lamellipodia formation (Oliferenko et al., 1999
; Oliferenko et al., 2000
). Furthermore, it has been shown that annexin II coimmunoprecipitates with constitutively active Rac1 and is suggested to be involved in trapping the diffusive Rac1 complexes in the plasma membrane, causing local activation of Rac1 (Hansen et al., 2002
). We have demonstrated that the association of the cytoplasmic tail of Na,K-β with annexin II is PI3-kinase dependent and that this binding is essential for the formation of lamellipodia and suppression of cell motility in MSV-MDCK cells. Because the cytoplasmic tail-deficient mutant of Na,K-β that fails to bind annexin II did not show increased Rac1 activity or suppression of motility, annexin II binding to Na,K-β is essential for the Na,K-β–mediated suppression of motility in MSV-MDCK cells.
How might Na,K-ATPase be involved in the suppression of cell motility? Based on our results, we propose the following model () to explain the mechanism of Na,K-β–mediated suppression of cell motility. Repletion of Na,K-β in MSV-MDCK cells increases the levels of Na,K-α expression (Rajasekaran, 2004). Elevated Na,K-α levels are involved in its increased binding to p85 leading to the activation of PI3-kinase (). PI3-kinase activation increases the levels of PIP3
(Welch et al., 2003
), which in turn activates Rac1 (; Welch et al., 2003
) as well as facilitates binding of annexin II to Na,K-β (). Inhibition of PI3-kinase by LY294002 abrogates Rac1 activation () and binding of annexin II to Na,K-β (). A complex containing Na,K-α, Na,K-β (), active PI3-kinase (as revealed by PH-Akt-GFP localization to the lamellipodia; ), and annexin II (Ghitescu et al., 2001
) localizes to the plasma membrane. Sequestration of active Rac1 into this complex by annexin II (Hansen et al., 2002
) leads to the formation of lamellipodia and suppression of cell motility. Because pharmacological inhibition of Na,K-ATPase does not prevent lamellipodia formation or Rac1 activation (), we suggest that protein–protein interactions via Na,K-α and Na,K-β, rather than ion transport function of Na,K-ATPase, are involved in the suppression of motility in MSV-MDCK cells. Moreover, because inhibition of PI3-kinase abolishes lamellipodia (), and suppression of cell motility (), Na,K-β–mediated suppression of cell motility in MSV-MDCK cells is dependent on the activation of PI3-kinase. Thus, these results demonstrate that Na,K-ATPase is a multifunctional protein, acting both as an enzyme as well as a signaling molecule involved in the regulation of cell motility in epithelial cells.
Figure 8. Model showing the mechanism of Na,K-β–mediated suppression of cell motility. 1) Repletion of Na,K-β in MSV-MDCK cells increases Na,K-α levels. 2) High levels of Na,K-α lead to the increased tyrosine phosphorylation (more ...)
Increased motility is a prerequisite for invasion and metastasis. Understanding molecular mechanisms involved in the regulation of cell motility in carcinoma cells should provide insights into novel therapeutic strategies to treat invasive and metastatic cancers. In this study, we have uncovered a novel role for Na,K-β in the suppression of cell motility in epithelial cells. We have shown that repletion of Na,K-β in MSV-MDCK cells significantly suppressed motility (Rajasekaran et al., 2001b
). Reexpression of E-cadherin also suppressed motility in these cells (Rajasekaran et al., 2001b
). However, coexpression of both these proteins suppressed the motility even further, indicating a synergistic role of these two proteins in the suppression of motility of carcinoma cells (Rajasekaran et al., 2001b
). Therefore, loss of either E-cadherin or Na,K-β or both in carcinoma cells might be associated with their increased motility and invasive behavior. Ability to increase the expression of both these proteins in carcinoma cells should have significant therapeutic value to impede cancer cell invasion and metastasis.