ABSTRACT
BACKGROUND: Both cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness. METHODS: NHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH. RESULTS: The cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression. CONCLUSION: NHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.
ABSTRACT
Upregulation of the Na(+)/H(+) exchanger isoform 1 (NHE1) has been correlated with tumor malignancy. In contrast, moesin-radixin-ezrin-like protein (merlin) is a tumor suppressor that protects from cancerogenesis. Merlin is highly related to the members of the ezrin, radixin, and moesin (ERM) protein family that are directly attached to and functionally linked with NHE1. In addition, merlin inhibits the MAPK cascade and the Rho-GTPases known to activate NHE1 activity. The present study investigates whether NHE1 expression and activity affect merlin or, conversely, whether merlin has an impact on NHE1 in human melanoma (MV3) cells. Indeed, features of merlin-deficient MV3 cells point to a functional link: merlin-deficient cells showed a decreased NHE1 expression and, paradoxically, an increase in NHE1 activity as measured upon cytosolic acidification (NH4Cl prepulse method). Loss of merlin also led to an elevated cell motility that could be further increased by NHE1 overexpression, whereas NHE1 overexpression alone had no effect on migration. In contrast, neither NHE1 expression nor its activity had an impact on merlin expression. These results suggest a novel tumor suppressor function of merlin in melanoma cells: the inhibition of the proto-oncogenic NHE1 activity, possibly including its downstream signaling pathways.