Processing of MIA protein during melanoma cell migration

Abstract

MIA (melanoma inhibitory activity) protein, identified as a small 11 kDa protein highly expressed and secreted by malignant melanoma cells, plays an important functional role in melanoma development, progression and tumor cell invasion. Recent data describe a direct interaction of MIA protein with cell adhesion receptors integrin α₄β₁ and integrin α₅β₁ and extracellular matrix molecules. By modulating integrin activity MIA protein mediates detachment of melanoma cells from surrounding structures resulting in enhanced invasive and migratory potential. However, until today a detailed understanding of the processes of MIA function is missing. In this study, we show that after binding of MIA protein to integrin α₅β₁, MIA protein is internalized together with this cell adhesion receptor at the cell rear. This mechanism enables tumor cells to migrate in a defined direction as appropriate for invasion processes. Treatment of melanoma cells with PKC-inhibitors strongly reduced internalization of MIA protein. Endocytosis is followed by dissociation of MIA–integrin complexes. In acidic vesicles MIA protein is degraded while integrins are recycled. Treatment of melanoma cells with MIA inhibitory peptides almost completely blocked the MIA protein uptake into cells. As MIA protein has a major contribution to the aggressive characteristics of malignant melanoma in particular to formation of metastasis, it is important to elucidate the MIA functional mechanism in tumor cells to find novel therapeutic strategies in the fight against skin cancer. © 2009 UICC

Malignant melanoma is characterized by aggressive local growth and early formation of metastasis, and accounts for 75 percent of deaths associated with skin cancer. Previously, melanoma inhibitory activity (MIA) has been identified as an 11 kDa protein strongly expressed and secreted by malignant melanoma cells but not expressed in melanocytes.1 Subsequent in vitro and in vivo experiments revealed that MIA protein plays an important functional role in melanoma development and cell invasion,2 hence MIA expression levels parallel closely the capability of melanoma cells to form metastases in syngeneic animals.3, 4 Increased MIA serum concentrations serve as a reliable clinical tumor marker to detect and monitor metastatic diseases in patients with malignant melanomas.1, 5, 6

The three-dimensional structure of the protein was solved by multidimensional nuclear magnetic resonance (NMR)7–9 and X-ray crystallography techniques.10 Corresponding data indicate that MIA defines a novel type of secreted protein: the MIA protein family, consisting of MIA and the homologous proteins OTOR, MIA-2 and TANGO (MIA-3). The MIA protein family is the first family of secreted proteins comprising an SH3 domain-like fold in solution.11 Furthermore, phage display experiments and NMR spectra revealed that MIA protein interacts with peptides matching to extracellular matrix proteins including human fibronectin type III repeats and laminin structures. In previous studies using far Western blotting and co-immunoprecipitation MIA protein was identified to bind to the cell surface proteins integrin α₄β₁ and integrin α₅β₁.12 Thus, MIA protein modulates integrin activity and thereby mediates detachment of cells from extracellular matrix proteins, resulting in enhanced invasive and migratory potential of melanoma cells.

In cell migration processes integrins, mediating cell–cell and cell–extracellular matrix contacts, undergo endocytic–exocytic transport. Adhesion receptor recycling is described as a process where at the cell rear integrins are internalized and subsequently transported within recycling vesicles to the leading edge of the migrating cell. Here, they are re-exocytosed to build new adhesion contacts to extracellular matrix molecules.13, 14 Now the question arises, how MIA protein contributes to migration and invasion after its secretion from tumor cells. This study elucidates the mechanism by which MIA protein promotes cell detachment and thus influences formation of cancer metastases. We found that extracellular MIA protein, directly binding to integrin α₅β₁, is internalized together with this cell adhesion receptor at the cell rear. This located uptake of MIA protein results in focal cell detachment at the rear cell pole and allows a directed migration. We also demonstrate that after MIA–integrin endocytosis, these receptor-MIA complexes dissociate and MIA protein is degraded in acidic vesicles. Treatment of melanoma cells with MIA-inhibitory peptides results in a dramatically decrease of MIA protein internalization in a dose dependant manner. As MIA protein promotes invasive behavior of malignant melanoma cells, it is necessary to find a mechanistic explanation for observed MIA effects to develop a novel therapeutic strategy.

Arf6, ADP-ribosylation factor 6; BIMI, bisindolylmaleimide I; BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified essential medium; DIEA, N,N-diisopropylethylamine; FITC, fluorescence-isothiocyanat; HOBt, hydroxybenzotriazole; MBHA, 4-methylbenzhydrylamine hydrochloride; MIA, melanoma inhibitory activity; MTOC, microtubule organizing center; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; PKC, protein kinase C; Rab11, member RAS oncogene family; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.

Material and methods

Cell lines and cell culture conditions

The melanoma cell line Mel Im, established from a human metastatic bioptic sample (generous gift from Dr. Johnson, University of Munich, Germany) was used in all experiments. Additionally, main experiments were also conducted using human cell lines Mel Ju, SK Mel 28 and A375, which were all derived from metastasis of malignant melanoma. Cells were maintained in DMEM (PAA Laboratories GmbH, Austria) supplemented with penicillin (400 U/ml), streptomycin (50 μg/ml), L-glutamine (300 μg/ml) and 10% fetal calf serum (Pan Biotech GmbH, Germany) and split in 1:5 ratio every 3 days.

Protein labeling

For the conjugation of the orange fluorescing cyanine dye Cy3, 0.11 mg MIA protein or 0.4 mg BSA, respectively, was dissolved in 1 ml sodium carbonate-sodium bicarbonate buffer (pH 9.3), added to the dye vial (Cy™3 Mono-Reactive Dye Pack, Amersham GE Healthcare, UK) and mixed thoroughly. The reaction was incubated at room temperature for 50 min before separation of protein from free dye using a Sephadex™ G-25 M PD-10 Desalting column (Amersham Pharmacia Biotech, Sweden). During elution 2 pink bands occurred; the faster moving band represents Cy3-labeled MIA protein and Cy3-labeled BSA, respectively. The procedure was designed to label protein to a final molar dye/protein ratio between 4 and 12.

The fluorescent Cy3 label does not affect binding properties of MIA protein, as deduced from Boyden Chamber invasion experiments, where Mel Im cells were treated with Cy3-labeled MIA protein and, in comparison, with unlabeled MIA protein (Supporting Information Fig. 1A).

Immunofluorescence assays

Melanoma cells (5 × 10⁵), Mel Im, Mel Ju, SK Mel28 and A375, respectively, were grown in a 4-well chamber slide in 500 μl DMEM and incubated with 35 μl of 4.5 μM Cy3-labeled MIA protein or BSA, respectively, for 90 min at 37°C and 5% CO₂. Afterwards, cells were washed and fixed using 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 15 min and permeabilized.15 After rinsing with PBS for 5 times, coverslips were mounted on slides using Hard Set Mounting Medium with DAPI (Vectashield, H-1500) and imaged using an Axio Imager Zeiss Z1 fluorescence microscope (Axiovision Rel. 4.6.3) equipped with an Axio Cam MR camera. Images were taken using 40× or 63× oil immersion lenses. For a better illustration in all pictures Cy3 staining is depicted in yellow. Conspicuous extracellular located yellow dots perceptible in images comprising MIA Cy3 staining are dye-artefacts.

For Golgi marker experiments cells were seeded and incubated for 24 hr before further treatment. During this time span the reorientation of the microtubule organizing center (MTOC), a comparatively slow process that can take several hours after migratory stimuli, is ensured. After fixation of cells with 4% paraformaldehyde in 0.1 M PBS, permeabilizing and blocking of nonspecific binding sites with blocking solution (1% BSA/PBS) for 1 hr at 4°C and rinsing was performed. Cells were incubated with primary antibody mouse anti-Golgi protein [58K 9] antibody (Abcam, UK) in concentrations of 1 μg/ml at 4°C for 2 hr. The amount of migrating cells was determined by counting 3 times 50 cells. Cells, which show the characteristic MTOC staining pattern at the cell front were evaluated as “migrating,” whereas nonpolarized cells that show homogenously distributed staining were counted as “nonmigrating.”

To illustrate colocalization of MIA protein with integrin α₅β₁, cells were incubated with a 1:60 dilution of mouse anti-human integrin β₁ [CD29] antibody (Chemicon International) or a 1:40 dilution of mouse anti-human integrin α₅ antibody (Chemicon Internatonal), respectively, at 4°C for 2 hr after Cy3-labeled MIA protein treatment, fixation with 4% paraformaldehyde in PBS, permeabilization and blocking of nonspecific binding with 1% BSA/PBS. To exclude nonspecific binding of target primary antibody due to Fc-binding or other protein–protein interactions we also used a mouse IgG isotype control antibody (Chemicon) (data not shown).

To generate MIA/Rab11 costaining, cells were treated with Cy3-labeled MIA protein, fixed with 4% paraformaldehyde in PBS, permeabilized and nonspecific binding sites were blocked using 1% BSA/PBS. Afterwards, cells were incubated with a 1:50 dilution of mouse anti-Rab 11 antibody (BD Bioscience Pharmingen) at 4°C for 2 hr. After rinsing with PBS for 5 times, cells were covered with a 1:30 dilution of the secondary antibody (FITC-conjugated polyclonal rabbit anti-mouse immunoglobulin, DakoCytomation) in PBS at 4°C for 1 hr. Afterwards, cells were washed with PBS and mounted with Hard Set Mounting Medium with DAPI (Vectashield, H-1500) or Hard Set Mounting Medium without DAPI (Vectashield, H-1400), respectively.

To selectively stain acidic lysosomes, Mel Im cells, grown on a 4-well chamber slide, were incubated with LysoTracker red DND99 (Molecular Probes, Invitrogen) in a concentration of 60 nM for 90 min at 37°C, 5% CO₂. Afterwards, cells were washed, fixed using 4% paraformaldehyde in 0.1 M PBS for 15 min and permeabilized. After rinsing with PBS for 5 times, cells were covered with blocking solution (1% BSA/PBS) for 1 hr at 4°C followed by incubation with an 1:20 dilution of primary antibody rabbit anti-MIA antibody (Biogenes, Berlin, Germany) for 2 hr at 4°C. After washing with PBS cells were incubated with a 1:30 dilution of the secondary antibody (FITC-conjugated swine anti-rabbit immunoglobulin, DakoCytomation). In case of simultaneously staining acidic lysosomes, LysoTracker green DND26 (Molecular Probes, Invitrogen) in a concentration of 600 nM was incubated together with Cy3-labeled MIA protein on Mel Im cells for 90 min at 37°C and 5% CO_2. Without fixation cells were washed with PBS and mounted using Hard Set Mounting Medium without DAPI (Vectashield, H-1400).

MIA inhibitory peptide and PKC inhibitors

For inhibition of MIA protein uptake, Mel Im, Mel Ju, SK Mel28 and A375 cells, respectively, together with Cy3-labeled MIA protein and the respective inhibitor were incubated for 90 min at 37°C and 5% CO_2. Inhibitors were used in several final concentrations. AR54 (sequence: NSLLVSFQPPRAR), a MIA binding peptide deduced from peptide FN14, which was previously identified in a phage display experiment,8 was synthesized on solid-phase using HOBt/TBTU/DIEA and Rink Amide MBHA resin and was used at concentrations of 0.3 μM, 0.5 μM and 2 μM. Its ability to block MIA function was tested using a Boyden Chamber invasion assay.2 AR54 at a final concentration of 1 μM was able to almost completely inhibit MIA function without affecting integrin activity, indicating that specific binding of AR54 to MIA protein anticipates MIA interaction to extracellular matrix molecules and integrins (Supporting Information Fig. 1B).

As a negative control cells were also treated with scrambled peptide AR5 (sequence: Gly-Gly-Ser-Gly-NH₂) in concentrations of 1 μM and 3 μM. In all cases Cy3-labelled MIA protein uptake was not affected by AR5 (data not shown).

Both PKCα inhibitors 3-(N-[Dimethylamino]propyl-3-indolyl)-4-(3-indolyl)maleimide3-[1-[3-(Dimethylamino)propyl]1H-indol-3-yl]-4-(1Hindol-3-yl)1H-pyrrole-2,5dione Bisindolyl-maleimide I (BIM I) and 12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole (GÖ6976) were used in a final concentration of 30 μM. As a control, cells were treated with DMSO.

Results

Integrin heterodimers are known to regulate cell adhesion and migration. The mechanism of integrin recycling by vesicular transport contributes to cell migration by internalizing integrins at the cell rear and thereby facilitates their detachment to surrounding structures.14

In previous studies, it has been shown that MIA protein directly interacts with integrin α₄β₁ and integrin α₅β₁. This binding leads to cell detachment by decreasing interactions between melanoma cells and extracellular matrix molecules via inactivation of integrins. We, therefore, hypothesized that MIA protein regulates migratory behavior of melanoma cells by modulating integrin activity. This theory is supported by the fact that MIA expression levels directly correlate with the ability of melanoma cells to form skin cancer metastases.3, 4

As shown in Figure 1 Mel Im melanoma cells, seeded in medium confluence and incubated with Cy3-labeled MIA protein, show a strong Cy3-fluorescence intensity in intracellular vesicles asymmetrically distributed at 1 cell pole. About 40–50% of seeded Mel Im cells are migrating; nearly all of them present this characteristic unidirectional MIA-Cy3 staining pattern. Cells treated with Cy3-labeled BSA protein as a negative control under the same experimental conditions did not show any fluorescence signal, indicating that the labeled BSA protein is not endocytosed (Fig. 1d). The same characteristic MIA protein uptake was also found in all other melanoma cell lines tested: Mel Ju, SK Mel 28 and A375 (Supporting Information Fig. 2).

Figure 1. Mel Im cells were treated with Cy3-labeled MIA protein and as a negative control with Cy3-labled BSA protein. (a) Migrating cells internalize MIA protein and show a strong Cy3-fluorescence intensity asymmetrically distributed at 1 cell pole as indicated by the white arrows. (b) DAPI. (c) Merge. (d) BSA Cy3 negative control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

As MIA protein specifically interferes with attachment of melanoma cells we recently performed a phage display screening experiment to identify potential MIA-inhibitory peptides. These peptides were investigated in attachment analysis in a Boyden Chamber model on their ability to affect MIA function.2 AR54, one of these peptides deduced from FN14 structure,8 was able to almost completely inhibit MIA function at concentrations of 1 μM without affecting integrin function (Supporting Information Fig. 1B). After treatment of Mel Im cells with AR54 the endocytosis of Cy3-labeled MIA protein was reduced in a dose dependent manner. AR54 at concentrations of 0.3 and 0.5 μM moderately decreases MIA protein endocytosis, whereas a concentration of 2 μM almost completely inhibit MIA uptake as shown in Figure 2. For all other melanoma cell lines tested we observed comparable results (Supporting Information Fig. 3).

Figure 2. Mel Im cells simultaneously were treated with Cy3-labeled MIA protein (a) and AR54, a MIA protein binding peptide, which was able to inhibit MIA function. AR54 was added in different concentrations. Final concentrations of 0.3 μM (b) and 0.5 μM (c) moderately decrease MIA protein endocytosis whereas concentrations of 2 μM (d) almost completely inhibit MIA protein uptake. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

To further elucidate the MIA protein function in migratory behavior of melanoma cells, we determined the direction of cell migration. Illustrated in Figure 3 are 2 independent examples (I and II) where the observed MIA fluorescence staining pattern argues for a coordinated MIA protein uptake located at 1 cell pole (Fig. 3a, I). As generally accepted, directed migration begins with cell polarization and it has been shown in previous studies that the microtubule organizing center (MTOC) and the Golgi apparatus are reoriented toward the leading edge of cells in wound-healing migration assays.16–18 For the determination of the migration direction of Mel Im cells, cells were incubated with Cy3-labeled MIA protein (Fig. 3a, I) and afterwards stained with a Golgi marker (mouse anti-Golgi protein [58K 9] antibody) (Fig. 3b, I). Fluorescence analysis shown in Figure 3 revealed the uptake of MIA protein containing vesicles at the cell rear, confirming our hypothesis that MIA protein is strongly involved in detachment processes of migrating cells. This mechanism enables tumor cells to migrate in a defined direction. Nonpolarized cells and thus nonmigrating cells, perceptible at the homogeneous green staining (Fig. 3b, II), do not show the characteristically distributed Cy3 fluorescence (Fig. 3a, II). Using the other melanoma cell lines, in migrating cells the same fluorescence staining pattern appears. A375 cells, similar to Mel Im cells, show a strong migratory ability compared to the other cell lines Mel Ju and SK Mel 28 (Supporting Information Fig. 4).

Figure 3. For determination of the direction of migration, Cy3-labeled MIA treated Mel Im cells were stained with a Golgi marker mouse anti-Golgi protein [58K 9] antibody. The uptake of Cy3-labeled MIA containing vesicles takes place at the rear part of the cell (a) since the location of the MTOC-Golgi apparatus is toward the leading edge of migrating cells (b). Nonpolarized cells and thus nonmigrating cells, perceptible at the homogeneous green staining, do not show the characteristic MIA fluorescence located at the cell rear. (c) DAPI. (d) Merge. I and II are representative examples of 2 independent experiments. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

The observation that the MIA vesicular staining appears at the cell rear and the fact that MIA protein specifically binds to integrin structures α₄β₁ and α₅β₁ prompted us to investigate whether MIA protein is internalized together with integrins after binding. Therefore, Mel Im cells were treated with Cy3-labeled MIA protein and stained with anti-integrin β₁ [CD29] antibody. As illustrated in Figure 4, fluorescence-labeled integrins are distributed all over the cell, whereas in close proximity to the cell membrane integrins appear to accumulate (Fig. 4b). Interestingly, at early stages of the endocytosis process close to the cell membrane, we observed that these integrins are colocalized with Cy3-labeled MIA protein, in particular at the cell rear (Fig. 4c). After staining with an anti-integrin α₅ antibody we observed a similar staining pattern (data not shown). The same results were found for the other melanoma cell lines investigated (Supporting Information Fig. 5). These findings are also supported by data presented in previous studies. Based on far Western blotting and co-immunoprecipitation assays12 we found a direct interaction of MIA protein with integrin α₄β₁ and integrin α₅β₁. Together these results suggest MIA protein to be internalized into the cell together with integrins α₅β₁ after binding to these cell adhesion molecules and thereby blocking formation of extracellular matrix contacts.

Figure 4. Mel Im cells were treated with Cy3-labeled MIA protein. After fixation cells were stained with anti integrin β₁ [CD29] antibody. (a) Cy3-labeled MIA protein is mainly located at 1 cell pole (b) Immunofluorescence-labeled integrins are distributed all over the cell and accumulate at or close to the cell membrane. (c) Merge. As indicated in the image section by the white arrows, in regions under the cytoplasmic membrane, we observed that integrin β₁ colocalizes with Cy3-labeled MIA protein, here depicted in red. This phenomenon was observed especially at the cell rear.

Nowadays, it is known that integrins are either internalized by clathrin-dependent mechanisms or by nonclathrin-dependent mechanisms.19, 20 In terms of the latter, integrins can enter caveolae followed by an internalization route that is regulated by protein kinase Cα and dynamin.21 Since we found that MIA protein uptake into cells that were treated with GÖ6976 (Fig. 5c), blocking PKCα and β, or BIMI (Fig. 5b), inhibiting PKCα, β and γ, was dramatically decreased (Fig. 5), our theory of integrin-mediated MIA protein uptake was further supported.

Figure 5. Mel Im cells were incubated simultaneously with Cy3-labeled MIA and PKC inhibitors. The DMSO control shows the unidirectional Cy3-labeled MIA protein staining at the cell rear (a). Treatment with PKCα, β and γ inhibitor BIM I (30 μM) (b) and PKCα and β inhibitor GÖ6976 (30 μM) (c), respectively, leads to a dramatically decrease in endocytosis of Cy3-labeled MIA protein. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

In the cytoplasm close to the nucleus, Cy3-labeled MIA protein shows no colocalization with integrins (Fig. 4c). Thus, we concluded that MIA–integrin complexes were dissociated after endocytosis and that the 2 proteins now were transported in different ways. As with other cycling receptors, integrin heterodimers internalize to early endosomes from which they can be either returned directly to the plasma membrane or further trafficked to the perinuclear recycling compartment before recycling through Rab11- and/or Arf6-dependent mechanisms.14, 22–24 In Figure 6, 2 independent examples (I and II) for Mel Im cells treated with Cy3-labeled MIA protein and stained with anti-Rab11 antibody are illustrated. The MIA protein internalization takes place at the rear cell pole (Fig. 6a) whereas the Rab11 staining, here depicted in red, is homogeneously distributed all over the cell (Fig. 6b). Since Cy3-labeled MIA protein and integrin transporter-protein Rab11 do not colocalize (Fig. 6d), our model of intracellular dissociation of endocytosed MIA-integrin complexes further was confirmed. Under the same experimental conditions all other melanoma cell lines show comparable results: Cy3-labeled MIA protein does not colocalize with integrin transporter-protein Rab11 (Supporting Information Fig. 6).

Figure 6. Mel Im cells were treated with Cy3-labeled MIA protein (a). After fixation, cells were incubated with anti-Rab11 antibody (b). After endocytosis of Cy3-labeled MIA protein it was cleaved from integrins. Thus, the integrin transporter-protein Rab11 depicted in red, mediating integrin recycling, does not colocalize with MIA protein. (c) DAPI. (d) Merge. I and II are examples of 2 independent experiments.

To elucidate the fate of internalized MIA protein we treated cells with Lysotracker red DND99, a chromophore which specifically stains red acidic vesicles in the cytoplasm of cells (Fig. 7b). Lysosomes are organelles containing digestive enzymes catalyzing hydrolysis of macromolecules like proteins, polysaccharides, lipids and nucleic acids. The membrane surrounding lysosomes allows the enzymes to work at a pH value of 4 to 5, where these enzymes achieve a high activity. Mel Im melanoma cells were incubated with unlabeled MIA protein. Afterwards, MIA staining was performed using a rabbit anti-MIA antibody. As shown in 2 independent examples I and II in Figure 7a, MIA protein distribution depicted in green is similar to that of Cy3-labeled MIA protein shown in previous figures: there is a targeted uptake of MIA protein detectable at the cell rear of migrating cells. As indicated by the white arrows, exactly in regions comprising assemblies of acidic compartments colored in red, there was no MIA-staining detectable, pointing to degradation of MIA protein inside lysosomes. This phenomenon of disappearance of MIA signals strongly contributes to our model of dissociation of MIA protein from integrins after internalization. To further confirm our hypothesis of MIA protein degradation, cells were also treated with LysoTracker green DND26 together with Cy3-labeled MIA protein. As displayed in Figure 8c, MIA protein colocalizes with acidic cell compartments in close proximity to the nucleus. Unlike to detection of MIA protein using an anti-MIA antibody shown in Figure 7, this continuous Cy3-fluorescence signal is still detectable inside cytoplasmic acidic vesicles after digestion of the protein at a pH range of pH 4 to 5. In summary, our results demonstrate that MIA protein is internalized into the cell together with integrins and that MIA-integrin binding is dissociated. In the next step, MIA protein is digested in acidic vesicles while integrins are recycled.

Figure 7. Mel Im cells were treated with unlabeled MIA protein and Lysotracker red DND99, a chromophore which specifically stains red acidic vesicles in the cytoplasm of cells. After fixation, MIA protein staining was performed using a rabbit anti-MIA antibody. (a) MIA protein distribution depicted in green is similar to that of Cy3-labeled MIA protein: there is a targeted MIA protein uptake detectable at the cell rear of migrating cells. As indicated by the white arrows, exactly in regions comprising assemblies of acidic compartments colored in red (b), there was no MIA-staining detectable. (c) DAPI. (d) Merge. I and II represent 2 independent examples.

Figure 8. Mel Im cells simultaneously were treated with Cy3-labeled MIA protein (a) and LysoTracker green DND26 staining acidic lysosomes (b). As illustrated in the merge picture (c), MIA protein colocalizes with acidic cell compartments in the cytoplasm at the centre of the cell close to the nucleus. Colocalization is depicted in red and in the image section it is also indicated by white arrows.

Discussion

In this study, we analyzed the mechanism by which MIA protein, expressed and secreted by malignant melanoma cells, contributes to alteration of migratory and invasive behavior of these tumor cells. In previous investigations it was shown that MIA protein binds to extracellular matrix molecules including fibronectin, laminin and tenascin.2 MIA protein was also described to directly interact with the cell adhesion molecules integrin α₄β₁ and integrin α₅β₁.12 As a result, matrix structures are masked by MIA protein and moreover, neoplastic melanocytes enhance their metastatic capability by specifically changing their attachment to surrounding extracellular matrix molecules and basement membranes.

Experimental data described in this study demonstrate that secreted MIA protein is internalized together with integrin α₅β₁ after directly binding to this adhesion receptor at the cell surface. We also demonstrate that endocytosis is followed by dissociation of MIA–integrin complexes. Afterwards, MIA protein is degraded in acidic vesicles.

A similar endocytosis mechanism was also described for vitronectin, a plasma protein which was also found in the extracellular matrix. Many functions have been characterized for vitronectin, including regulation of the activity of both thrombin and plasminogen activator, as well as modulating the membrane attack complex of complement. Vitronectin comprises an Arg-Gly-Asp (RGD) sequence25 that can bind to either the α_vβ₃ or the α_vβ₅ integrin receptor.26, 27 Similar to MIA protein, vitronectin also mediates cell adhesion by this interaction. As a special feature for internalization of vitronectin together with the cell adhesion receptor, the interaction of both the α_vβ₅ integrin and a species of heparan sulfate proteoglycan are required. Binding to the extracellular matrix is a prerequisite for endocytosis of vitronectin deduced by the observation that multimeric vitronectin does not appear to be degraded from the fluid phase. Identical to what we observed for MIA protein, receptor-mediated endocytosis is followed by subsequent degradation of vitronectin in lysosomes. Further, it was demonstrated that effectors of protein kinase C, involved in signaling pathways between transmembrane signaling receptors, modulate vitronectin degradation by regulating the internalization.26 The inhibition of receptor mediated endocytosis of MIA protein in cells treated with protein kinase C inhibitors BIMI and GÖ6976 contributes to our hypothesis that MIA protein internalization may be regulated by a similar mechanism. Unlike vitronectin, MIA protein is bound to the cell surface receptor integrin α₅β₁ before internalization. In previous studies, it was reported that remodeling of matrix structures occurs via internalization of extracellular matrix proteins and degradation in lysosomes.28–31 It was shown that—identical to MIA protein—turnover of extracellular matrix protein fibronectin is processed via integrin α₅β₁ internalization. This endocytosis mechanism is constitutively regulated by caveolin-1 and can occur in presence or absence of fibronectin and fibronectin matrix.32 Not all fibronectin binding integrins can promote fibronectin endocytosis. Of the integrins tested, only α₅β₁ integrin was shown to participate in fibronectin endocytosis. Identical to our results for MIA protein it was also demonstrated that matrix turnover of fibronectin is followed by lysosomal degradation.33

Further, the prevention of MIA protein internalization after treatment of cells with peptides deduced from extracellular matrix proteins and integrin structures is consistent with our proposed mechanism. Before initial binding to integrin receptors, MIA protein was captured by these peptides. Next to their canonical role in physical adhesion of cells, interactions between cell surface molecules and matrix components provide pivotal contributions to a broad range of cellular processes in melanocytic cells. Thus, active detachment of melanoma cells induced by MIA protein may also be implicated in regulation of migration, apoptosis, secretion of proteases or matrix proteins and cell growth.34–38 It is known that such interactions between melanocytic cells and extracellular matrix involve foremost binding of integrins to specific epitopes within fibronectin and depend, to a significant extent, on activation of integrin α₄β₁ and integrin α₅β₁.39 Detachment from surrounding matrix structures is a basic requirement for melanoma cells to migrate, invade and finally metastasize in a systemic disease. To impair formation of metastases and control malignant melanoma metastases at the invasive state it is necessary to anticipate MIA binding to integrins and extracellular matrix molecules. Previously published data provide first evidence for a reduction of tumor size after application of 2 fibronectin-deduced peptides in a mouse melanoma model.2

In summary, our results demonstrate that MIA protein, binding to integrins and thus promoting detachment of cells from extracellular matrix structures, is internalized into the cell together with these cell adhesion receptors at the cell rear. MIA-integrin binding dissociates and in the next step, MIA protein is digested in acidic vesicles while integrins are recycled. Prevention of MIA protein internalization by capturing the protein by inhibitors in vivo may provide a novel therapeutic strategy for therapy of patients suffering from malignant melanoma.

Acknowledgements

The authors thank Dr. Johnson (University of Munich, Germany) for providing melanoma cell lines, Mr. Alexander Riechers for synthesis of MIA protein inhibitory peptides, Ms. Andrea Sassen and Ms. Marietta Bock for technical assistance.