MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells
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.
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.
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.
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.
KeywordsCell migration (Cortical) cell stiffness Matrix digestion Metastasis pH
murine melanoma cell line
ezrin, radixin, moesin protein family
cariporide (NHE1 inhibitor)
human melanoma cell line
Na+/H+ exchanger 1
N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid
phosphate buffered saline
sodium dodecyl sulfate
Tris-buffered saline with Tween20
Upregulation of the expression/activity of the ubiquitously expressed acid-extruding membrane transporter NHE1 (Na+/H+ exchanger 1) has been commonly correlated with tumor malignancy . NHE1, by taking advantage of the inwardly directed Na+-gradient across the plasma membrane, exchanges one H+ for one Na+ and thus contributes significantly to cellular pH homeostasis . A higher basal NHE1 activity is characteristic of tumor cells. It often leads to an increase in cytosolic steady state pHi, and an alkaline pHi favors (aerobic) glycolytic metabolism [3, 4], proliferation and evasion of apoptosis . The resulting decrease in extracellular pH (pHe), especially when accompanied by local intracellular alkalization , promotes tumor cell migration and invasion . The intracellular pH affects cytoskeletal dynamics while the pH at the cell surface modulates cell/matrix interactions and stimulates the activity of matrix metalloproteinases (MMPs) . Thus, by regulating pHi and pHe, NHE1 activity has a significant effect on the three major variables underlying cell motility: (i) MMPs clearing the way through the extracellular matrix, (ii) focal adhesion complexes ensuring a well-balanced substrate grip, and (iii) the cytoskeletal machinery including actomyosin dynamics considered as the engine for cell migration.
In addition to its pH- and osmoregulatory transport function, NHE1 (i) operates as a plasma membrane scaffold in the assembly of signaling complexes and (ii) serves as a structural anchor for actin filaments through its direct binding of actin binding proteins of the ezrin, radixin and moesin (ERM) family . As an actin anchoring protein, NHE1 maintains the cell shape by tying the plasma membrane to cortical actin filaments . H+ export and actin anchoring, both mediated by NHE1, dynamically coordinate the remodeling of actin and cell-substrate adhesion, which considerably contributes to cell motility .
The concerted action of actin and its accessory and regulatory proteins such as non-muscle myosin II is required not only for cell migration and invasion, but also for defining and modulating the cell shape . Accordingly, the cortical actin-myosin network, located right underneath the plasma membrane, determines the mechanical properties of the cell surface. Structure, density and integrity of the cortical actin meshwork determine cortical elasticity or stiffness  and can be affected by various parameters including the plasma membrane potential .
Local rupture or regulated tapering of the cortical actin network can lead to the formation of blebs. Usually, extensive blebbing indicates apoptosis, but the formation of blebs can also be characteristic of an amoeboid mode of cell migration, particularly of an invasive behavior in a spatially constrained environment [15, 16]. In this setting, blebs seem to be advantageous and replace lamellipodia and other protruding structures . Consistently with the occurrence of blebbing due to local changes in the cortical actin network during amoeboid movement in a dense extracellular matrix network, the stiffness of both tumor cells obtained from patients and cancer cell lines has been shown to inversely correlate with the invasion of three-dimensional basement membranes. Cancer cells with the lowest invasive potential are five times stiffer than those with the highest invasive potential . This holds true also for ovarian cancer cells that are generally softer than non-malignant ovarian epithelial cells  and for cancerous human bladder cells .
As NHE1 is able to tie the cortical actin to the membrane, and the integrity of the cortical actin network affects stiffness, blebbing and invasiveness, the present study aims to investigate a possible relationship between NHE1 expression, cell stiffness and invasiveness.
Material and methods
Human melanoma cells of the MV3 cell line , stably transfected with an empty pcDNA3 vector (control) or with the pcDNA3 vector carrying NHE1 (NHE1 overexpressing; ), were grown in bicarbonate buffered Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, Taufkirchen, Germany) supplemented with 10% (v/v) fetal calf serum (FCS) at 37 °C in a humidified atmosphere of 5% CO2, 95% air. The culture medium contained 0.6 g l−1 geneticin (G-418-sulfate; PAA Laboratories, Pasching, Austria) in order to select the transfected cells.
Detection of NHE1 and MMP3 by Western blot
Confluent cell cultures were washed with cold Dulbecco’s phosphate-buffered saline (PBS w/o Ca2+, Mg2+; Sigma-Aldrich) and lysed at 4 °C in radioimmunoprecipitation assay (RIPA) lysis buffer (150 mmol l−1 NaCl, 25 mmol l−1 Tris HCl (pH 7.6), 1% Nonidet P-40, 0.1% SDS, 1.0% sodium deoxycholate, a protease and a phosphatase inhibitor cocktail (cOmplete, Mini; PhosSTOP; both from Roche)). Lysates were scraped off and spun down at 13,000×g and 4 °C for 10 min. Protein concentrations were determined with the Bicinchoninic Acid Protein Assay Kit (Thermo Scientific). Equal amounts of protein (~ 30 µg) mixed with sample buffer (4:1 (v/v); 500 mmol l−1 Tris, 100 mmol l−1 dithiothreitol, 8.5% SDS, 27.5% sucrose, and 0.03% bromphenol blue indicator) were loaded, separated by SDS-PAGE (7.5% acrylamide gels; Minigel System, Bio-Rad Laboratories) and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon Transfer Membranes, Millipore) by tank blotting at 4 °C overnight. PVDF membranes carrying the blotted proteins were immersed in 5% (w/v) skim milk in 0.05% (v/v) Tween in PBS Dulbecco (w/o Mg2+; Biochrom AG) for 30 min at room temperature followed by overnight incubation with the primary antibody against NHE1 (mouse, 1:1000 in 5% skim milk/0.05% PBS-T; BD Biosciences). After washing (3 × 10 min in 0.05% Tween in PBS), blots were incubated for 1 h with a peroxidase (POD)-conjugated secondary antibody (goat anti-mouse POD, 1:25,000 in 5% skim milk/0.05% PBS-T, Dianova) and then washed again (3 × 10 min in 0.05% Tween in PBS). Blots were developed using a chemiluminescence kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Autoluminography was carried out with a ChemiDoc XRS gel documentation system and Quantity-One analysis software (Bio-Rad Laboratories). To control protein loading, membranes were stripped and then probed with a monoclonal anti-β-actin antibody (anti-mouse, 1:10,000; Sigma Life Science; secondary antibody 1:25,000 (goat anti-mouse POD, Dianova)). The Quantity-One software (Bio-Rad) was applied for densitometric analyses. The protein amount was normalized to the amount of β-actin.
Confluent cell cultures were thoroughly washed with PBS (Dulbecco, Biochrom AG) and then kept in serum-free, G418-containing RPMI 1640 medium for 24 h. In order to inhibit NHE1 activity, cariporide (HOE642; Santa Cruz Biotechnology; final concentration 10 µmol l−1) was added and renewed after 12 h. After 24 h, 1 ml of the MMP-containing medium was used for trichloroacetic acid (TCA) precipitation. After adding 225 µl 60% TCA (final concentration ~ 12%) and 139 µl 1%Triton (final concentration ~ 0.1%), the medium was vortexed and incubated on ice for 20 min. The precipitate was spun down at 14,000 rpm, 4 °C for 20 min, and washed twice with EtOH for 30 min. Each washing step was followed by 20 min centrifugation. Finally, the pellet was rinsed with ice cold acetone, centrifuged for 20 min, dried at 37 °C, resuspended and vortexed in sample buffer (2% SDS, 10% Glycerol, 60 mmol l−1 Tris–HCl (pH6.8), 0.001% bromophenol blue, 5% β-mercaptoethanol), and then heated to 95 °C for 5 min. 40 µl samples were subjected to electrophoresis in 4.5–15% SDS–polyacrylamide gradient-gels run at 30 mA. The separated proteins were electrotransferred to a nitrocellulose membrane (Protran nitrocellulose transfer membrane, BA 83, 0.2 µm, Whatman plc) at a constant current of 80 mA for 3 h at 4 °C. Total protein detection served as a loading control and was performed using the Pierce MemCode™ reversible protein stain kit (Pierce Biotechnology Inc.). The nitrocellulose membrane was destained using the MemCode stain eraser. Unspecific binding sites were blocked with Tris-buffered saline (TBS-T: 50 mmol l−1 Tris/HCl (pH7,4), 150 mmol l−1 NaCl, 0.05% Tween-20) containing 5% skim milk and 1% bovine serum albumin for 1 h at room temperature. The nitrocellulose membrane was then incubated with a primary antibody against MMP3 (monoclonal from rabbit (Abcam), 1:1000 in TBS-T containing 2.5% skim milk and 0.5% BSA) at 4 °C overnight. The nitrocellulose membrane was rinsed with TBS-T (3 × 15 min) followed by 1 h incubation with the secondary, horseradish peroxidase-conjugated antibody (donkey anti-rabbit (Amersham BioSciences), 1:10,000 in TBS-T with 2.5% skim milk and 0.5% BSA) at room temperature. After washing with TBS-T (3×), the membrane was developed employing the SuperSignal West Femto Maximum Substrate (Thermo Fisher Scientific). Detection of labeled protein bands was performed with a chemiluminescence imaging system (Fusion SL4.2P, Vilber Lourmat).
Cells were seeded onto collagen type I-coated (Collagen G, Biochrom AG; final concentration 0.4 mg ml−1) coverslips and cultured for 2 h. The cells were then fixed with 3.5% paraformaldehyde (w/v) in PBS (Dulbecco, Biochrom AG) for 30 min and permeabilized for 25 min in 0.1% (v/v) Triton X-100/TBS in order to ensure that the intracellular F-actin epitopes were accessible to the antibody. After washing with PBS (2×), nonspecific binding sites were blocked with 3% bovine serum albumin (BSA) in PBS (w/v) for 2 h at room temperature. The cells were then stained with Alexa Fluor® 488 Phalloidin (Invitrogen AG; dilution 1:100) for 45 min. Prior to Dako mounting (Dako A/S, Glostrup, Denmark), the cells were washed in PBS once again. Images were taken with a digital camera (Model 9.0, RT-SE-Spot, Visitron Systems, Puchheim, Germany) fitted to an inverted microscope (Axiovert 200, Carl Zeiss AG) and controlled by MetaVue software (Visitron Systems).
Measuring cell stiffness by atomic force microscopy (AFM)
Employing a JPK NanoWizard 3 (JPK Instruments, Berlin, Germany) combined with a Leica DMI 6000 (CS Trino AFC) inverted fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) and the JPK SPM software (JPK Instruments) both the cortical and the bulk stiffness of the two MV3 clones were measured. To this end MV3 cells were seeded onto collagen type I (Collagen G, Biochrom AG)-coated glass bottom dishes (35 mm in diameter, WillCo Wells) at a density corresponding to ~ 70% confluency and were kept in HEPES buffer (122.5 mmol l−1 NaCl, 5.4 mmol l−1 KCl, 0.8 mmol l−1 MgCl2, 1.2 mmol l−1 CaCl2, 5.5 mmol l−1 glucose, 10.0 mmol l−1 HEPES; pH 7.4) at 37 °C in a heated chamber mounted on the stage of the microscope. For the AFM-measurements a soft cantilever (nominal spring constant = 0.03 N m−1, Novascan Technologies) with a spherical tip (sphere diameter = 10 µm) and a maximum loading force of approximately 1 nN were used.
Essential mechanical probing parameters including deflection sensitivity and cantilever spring constant were calibrated prior to each experiment. Cell stiffness was chosen as a preferred quantitative readout of mechanical alteration of the cells. The stiffness value provides the most direct and straightforward mechanical readout for a given combination of an AFM probe and a sample. On the other hand, such precise calculation of the elastic modulus relies heavily on several additional parameters  which could not be obtained during our experiments. Cell nanoindentation was performed by applying a maximal loading force of 1 nN at a loading rate of 1 µm s−1. Resulting cell deformation was used to calculate the cell stiffness as described previously .
Preparation of collagen matrices for cell migration
A collagen I substrate was prepared by gently mixing 210 µl of 5× RPMI1640, 210 µl of 5× HEPES-buffer (final concentration in the polymerized collagen gels: 10 mmol l−1), 245 µl of distilled water and 430 µl of Collagen G (containing acid-soluble calfskin collagen type I at a concentration of ~ 4 mg ml−1). The pH value of the mixture was adjusted to 7.4 with 1 M NaOH. A thin layer (~ 200 µl) of this collagen I matrix polymerized on the bottom of a 12.5 cm2 culture flask (Falcon, Corning Inc.) in a humidified atmosphere at 37 °C overnight. The next day, the cells were seeded onto this matrix and allowed to attach and spread for 6 h.
In one set of experiments matrix metalloprotease (MMP) activity was disabled by fixing the matrix with 2 ml of 2% glutaraldehyde in PBS (v/v) for 15 min. The matrix was washed (5 × 5 min) with PBS and stored in PBS at 4 °C overnight. The next day, the fixed matrix was washed again (5 × 5 min PBS) and the cells were seeded.
Transmigration was determined employing Boyden chamber assays. 20 µl of the collagen I mixture (composition as described above) were allowed to polymerize on a filter-membrane (insert for a 24 well plate, 8.0 µm pore size; ThinCert, Greiner Bio-One GmbH) at 37 °C in a humidified atmosphere overnight. 200,000 cells per filter were seeded onto this collagen matrix. After 24 h incubation in RPMI1640 with G-418 and serum, the medium was gently renewed for another 24 h. Cells were then fixed and stained with crystal violet (Sigma-Aldrich) in PBS. The matrix and the remaining cells on the upper side of the filter were removed and excess crystal violet was washed away with PBS. The invasive cells that remained on the lower side of the filter and those on the bottom of the well were counted.
MMP activity was inhibited by 10 µmol l−1 NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid; Sigma-Aldrich), and NHE1 was inhibited with 10 µmol l−1 cariporide (HOE642). DMSO, the solvent for NNGH and cariporide, reached a final concentration of 0.1%.
Total MMP activity was disabled by fixing the matrix with 1 ml of 2% glutaraldehyde in PBS (v/v) for 15 min. In order to ascertain the role of actin in transmigration through a fixed matrix, cells were exposed to Cytochalasin D (50 nmol l−1) over the entire experiment (48 h).
Matrix digestion in situ
20 µl of the collagen mixture (see above) were allowed to polymerize on coverslips (ø 15 mm, R. Langenbrinck GmbH, Germany) for at least 3 h in a humidified atmosphere (5% CO2, 95% air) at 37 °C. The matrices were then either kept in PBS at 4 °C until use, or they were fixed with 2% glutaraldehyde in PBS (v/v) for 15 min, washed (5 × 5 min) with PBS and stored in PBS at 4 °C overnight. The next day, the fixed collagen substrates were washed again (5 × 5 min PBS), and cells were seeded onto the fixed and the native matrices. The medium was renewed after 24 h. After 48 h the cells on the native matrix were fixed with 2% glutaraldehyde in PBS (v/v) for 15 min and then washed with PBS (5 × 5 min). The coverslips carrying the cells were mounted on glass slides with fluorescence mounting medium containing DAPI (4′,6-diamidino-2-phenylindol; Dako A/S, Glostrup, Denmark). The glutaraldehyde-induced autofluorescence of the collagen substrate (at 488 nm) was evaluated employing the same setup as that used for the actin staining. The NIH ImageJ software (http://rsb.info.nih.gov/ij/) was used to determine the fluorescence intensity per visual field (233.086 pixels).
20 µl of conditioned media from confluent cell cultures were mixed with equal volumes (20 µl) of twofold concentrated sample loading buffer (2 mmol l−1 EDTA, 2% SDS, 20% glycerol, 0.02% bromophenol blue, 20 mmol l−1 Tris/HCl, pH 8.0) and subjected to electrophoresis on a 1% gelatin-(porcine skin, Sigma-Aldrich) containing 4.5–15% gradient SDS–polyacrylamide gel run at 40 mA. The gel was washed twice for 30 min in 2.5% Triton-X 100, rinsed in distilled water and then developed in 50 mmol l−1 Tris/HCl (pH 8.5) containing 5 mmol l−1 CaCl2 overnight at 37%. It was stained with Coomassie brilliant blue R250 (0.15% Coomassie BB R-250; Bio-Rad Laboratories).
Data are presented as the mean values ± SEM. Depending on the experiment type experiments were repeated three up to nine times. Significance of the data was determined with the student’s unpaired or paired t test. p < 0.05 was set as the level of significance (*p < 0.05; **p < 0.01; ***p < 0.001).
Expression of NHE1
Cortical stiffness depends on NHE1 expression
NHE1 expression affects F-actin
NHE1 expression affects matrix digestion
NHE1 overexpression reduces cell migration on fixed collagen type I
Effects of matrix fixation and NHE1 expression on cell migration and morphology
Mock control (n = 30; N = 5)
Overexpressed (n = 40; N = 3)
Mock control (n = 40; N = 3)
Overexpressed (n = 41; N = 3)
Speed [µm min−1]
0.44 ± 0.03
0.034 ± 0.02
0.48 ± 0.04
0.13 ± 0.01
Net distance [µm]
61.92 ± 9.42
45.17 ± 5.6
57.92 ± 9.07
11.93 ± 2.33
Total distance [µm]
130.92 ± 8.61
103.12 ± 5.78
143.37 ± 10.67
37.84 ± 2.83
0.45 ± 0.05
0.44 ± 0.04
0.38 ± 0.04
0.28 ± 0.04
0.43 ± 0.03
0.5 ± 0.02
0.3 ± 0.01
0.42 ± 0.02
888 ± 112
1115 ± 84
1251 ± 78
1298 ± 94
NHE1 overexpression fuels invasion of native collagen type I
NHE1 expression stimulates secretion of MMP3
Because MMP3 activates the collagenases MMP1, MMP8 and MMP13  that are capable of degrading collagen types I, II, III, V and IX as well as native fibrillary collagen  which is crucial for melanoma metastasis, we tested for MMP3 secretion by performing western blot analyses (Fig. 9b). NHE1 overexpressing cells, as opposed to the mock control, secreted a substantial amount of MMP3. MMP3 secretion was reduced in cariporide (HOE642)-treated cells, indicating that both NHE1 expression and its activity promote MMP3 secretion.
The main purpose of the present study was to check a possible relationship between NHE1 expression, the cortical cell stiffness of human melanoma (MV3) cells and their ability to invade a defined collagen I substrate. We found that the overexpression of NHE1 leads to an increase in cortical stiffness without affecting the bulk stiffness (Fig. 2). This increase in cortical stiffness is accompanied, if not even caused, by a rearrangement of cortical F-actin (Fig. 3). At the same time, although showing an increase in cortical stiffness, the NHE1 overexpressing cells are significantly more invasive (Fig. 7). This increase in invasiveness is probably mediated by an elevated MMP3 secretion and activity (Figs. 8, 9). Since NHE1, especially its activity, stimulates not only the expression of several MMPs at both mRNA and protein level but also their pH-dependent activity [32, 33, 34], we minimized the effects of NHE1-mediated MMP activity by utilizing aldehyde-fixed matrices. The cells are hardly able to invade such a fixed collagen substrate. In those cells that do transmigrate across the fixed substrate, the NHE1 inhibitor cariporide (HOE642) has no effect suggesting that NHE1 activity-dependent processes are not crucial under these conditions (Fig. 7). Sound actin dynamics, however, are necessary, as cytochalasin D, an inhibitor of actin polymerization, impedes transmigration across a fixed substrate almost completely.
In the present migration experiments, NHE1 overexpression causes significant cell rounding and slowdown (Figs. 5, 6). The observation that cell rounding accompanied by a decrease in 2D motility correlates with an increased invasiveness is consistent with the finding that murine melanoma (B16V) cells spread and migrate on a basement membrane-like matrix, whereas they hardly migrate on, and instead invade, a dermis-like matrix .
NHE1 acts as a structural anchor for actin filaments by directly binding actin binding proteins of the ERM family . Usually, an N-terminal domain of an activated, i.e. phosphorylated, ERM protein binds to a positively charged residue in the cytoplasmic tail of a transmembrane protein, such as NHE1, while its C-terminal domain binds actin filament(s). Thus, ERM proteins cross-link the plasma membrane to the underlying cortical actin . One member of the ERM family is moesin . The activation of moesin upon entry into mitosis is required for cell rounding accompanied by an increase in cortical rigidity . This finding is in line with the present observation that an overexpression of NHE1, one of the binding partners of the ERM family, causes an increase in cortical stiffness associated with a rearrangement of the cortical F-actin.
In addition to its function as a mere structural element, NHE1 may affect cortical stiffness also by its activity. Comparing the cortical stiffness of completely untreated NHE1 overexpressing MV3 cells with that of cariporide-treated NHE1 overexpressing cells does not reveal a significant difference. However, the cortical stiffness of cells treated with the solvent DMSO alone is slightly higher than that of cells treated with cariporide solved in DMSO. Without any doubt, DMSO has a strong impact on the plasma membrane. It induces membrane thinning, increases the fluidity of the membrane’s hydrophobic core and, at higher concentrations, creates transient water pores in the membrane . DMSO as a component of freezing media causes a significant increase in the stiffness of mouse embryonic fibroblasts . Moreover, DMSO increases the membrane permeability for K+ in a dose-dependent manner in monocytes (THP-1 cells ). The diameter of a hydrated K+ ion is assumed to be ~ 0.133 nm while that of a hydrated Na+ ion comes to ~ 0.5 nm (0.095 nm non-hydrated ionic radius + ~ 0.4 nm hydration shell ). Nonetheless, it could be possible that DMSO increases the membrane permeability for Na+ as well. The DMSO-mediated increase in the membrane permeability for water and/or Na+ would lead to an osmotic swelling resulting in a higher stiffness. Provided that unfolding membrane reservoirs are not available, osmotic swelling would cause the cell membrane to stiffen  while, at the same time, the cortical actin cytoskeleton would behave like an expanding sponge and enhance the stiffening . In the present study, cariporide (HOE642) in presence of its solvent DMSO reduces the cortical stiffness. At this point, we are not able to determine to what extent (i) a decrease in the number of Na+ ions imported by NHE1 and thus osmotic shrinkage [45, 46], (ii) a decrease in cytosolic pH  or (iii) other factors such as pH-dependent signaling  contribute to the cariporide-induced, slight decrease in cortical stiffness.
The restructuring of the cortical actin meshwork accompanied by an increase in cortical stiffness (Figs. 2, 3) most likely modulates both the architecture including the lipid packing  and the composition of the plasma membrane  of NHE1 overexpressing cells. This could then modulate enzymatic activities  and catalyze the conversion of sphingolipids such as ceramide, sphingomyelins or glycosphingolipids [52, 53]. For instance, the acid sphingomyelinase catalyzes the cleavage of sphingomyelin to produce ceramide and phosphorylcholine, and the sphingomyelin deacylase catalyzes the hydrolysis of N-acyl-sphingosylphosporylcholine leading to the generation of a fatty acid and sphingosylphosphorylcholine (SPC). SPC induces the expression and secretion of MMP3 . In the present Western blot analysis, NHE1 overexpression in MV3 cells is accompanied by an increase in MMP3 secretion. Normally, at physiological pH values of ~ 7.4, NHE1 activity and thus the number of H+ delivered to the cell surface are rather low. However, not only the acid sphingomyelinase  but also the sphingomyelin deacylase  show their maximum activities at rather low pH values of pH ~ 5.0. Since we found an increased MMP3 secretion in the NHE1 overexpressing cells and because in a wide variety of cancers including melanoma cells NHE1 activity is considerably elevated [1, 3, 57, 58], it is conceivable that the number of H+ ions released at the cell surface may be high enough to sufficiently stimulate the sphingomyelin-converting enzymes (and through SPC indirectly MMP3) even though neither the proper nor a large-area pH optimum is reached. Immunoblotting the media revealed that NHE1 overexpressing MV3 cells do secrete more MMP3 depending on NHE1 activity (Fig. 9b). Furthermore, MMP3 as activator of collagenases  most likely plays a crucial role because MMP inhibition by NNGH leads to a distinct decrease in transmigration across native matrices (Fig. 8).
Both expression and activity of NHE1 affect the cell cycle and correlate with proliferation [59, 60]. Therefore, the increase in the number of MV3 cells that invade native matrices could be partially due to an increased proliferation associated with NHE1 overexpression (Fig. 7a). On the other hand, this assumption should hold true also for NHE1 overexpressing cells invading a fixed matrix. But there is no difference in transmigration between NHE1 overexpressing and control cells.
Fixation of extracellular matrices with glutaraldehyde modifies their micro-elastic properties and leads to a substantial increase in matrix stiffness . The matrix stiffness modulates cell behavior , induces malignant phenotypes  and can trigger epithelial–mesenchymal transition (EMT ). In fact, the MV3 cells are more spread and less spherical on a glutaraldehyde-fixed compared to a native collagen type I substrate. While unchanged in control cells, cell motility of NHE1 overexpressing MV3 cells is significantly decreased on a fixed substrate (Fig. 5). Albeit this is probably caused by the absence of H+-dependent events such as modulation of pH-sensitive cell/matrix interactions  and MMP activity , a certain impact of the matrix rigidity on cell motility cannot at all be excluded and could also affect transmigration across the fixed substrate. In a three-dimensional setting such as the Boyden chamber/transmigration assay it is hardly possible to precisely dissect to what extent the different physiological and biophysical parameters affect invasiveness.
Given the entire data set, we conclude that in MV3 cells, NHE1 overexpression causes a rearrangement of F-actin at the cell cortex associated with an increase in cortical stiffness. This rearrangement in F-actin is likely to rely 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 on a collagen type I substrate, most likely due to increases in MMP3 secretion and activity. This is in line with the proposed concerted roles of cortactin and F-actin in regulating the secretion of proteases . In addition, the link between NHE1 overexpression and MMP3 secretion may also include the architecture and composition of the plasma membrane and should be analyzed in future studies. In the long term and with simultaneous consideration of the (cellular) lipid metabolism, the relationship between the expressions of NHE1 and MMP3 may gain in importance for the development of anticancer drugs.
The authors would like to thank Dr. Jochen Seebach and Prof. Dr. Hans-Joachim Schnittler from the Institute of Anatomy and Vascular Biology in Münster, Germany, for helping with optimizing the matrix fixation protocol. This work was supported by a German Cancer Aid Grant (#111262) to CS.
FF generated the cell clones and delivered the fundamental idea. DK designed and performed the vast majority of (all types of) experiments. IL and VH supervised and performed AFM experiments. SN performed migration assays and actin staining. RD designed, supervised and performed zymography assays and MMP blots. CS designed and supervised experiments and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by a German Cancer Aid Grant (#111262) to CS.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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