MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells

Abstract

BackgroundBoth 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.MethodsNHE1 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.ResultsThe 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.ConclusionNHE1, 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.

The ACE Inhibitor Lisinopril Stimulates Melanoma Cell Invasiveness by Inducing MMP2 Secretion

“…However, in MV3 cells, we did not detect MMP9, neither at mRNA nor at protein level, which is consistent with a previous analysis of MV3 cells' MMP expression [83]. Besides, although MMP3 has been shown to potentially contribute to MV3 cell invasion [84], the ARP-100 concentration of 100 nmol l -1 used in the present study is too low to significantly affect MMP3. We therefore conclude that here ARP-100 inhibited MMP2 only.…”

Advances in research on the regulatory mechanism of NHE1 in tumors (Review)

“…This rearrangement in F-actin likely relies on the pH-sensitive and, thus, NHE1-dependent interaction between cortactin and cofilin. Despite their higher cortical stiffness, NHE1-overexpressing cells are considerably more invasive in a collagen type I substrate, which is most likely due to increases in MMP-3 secretion and activity (20). In hypoxic cancer cells, NHE1 localizes to invasive podocytes, where it promotes the formation of invasive pseudopods by expressing and activating p90 ribosomal S6 kinase (p90RSK) (21).…”

BEX1 and BEX4 Induce GBM Progression through Regulation of Actin Polymerization and Activation of YAP/TAZ Signaling

“…Actin filaments, along with microtubules, function as the core cytoskeleton and regulate the motility of tumor cells through the formation of the principal structure of filopodia [ 30 ]. Although the inhibition of cytoskeleton formation could be life-threatening, the use of inhibitors for F-actin formation, such as jasplakinolide, cytochalasin D, and Lat A, has previously been identified as a promising strategy to suppress tumor malignancy [ 31 , 32 , 33 ]. Given these observations, it has remarkable potential and further studies are needed to evaluate the safety of Lat B.…”