Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor

Abstract

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on alphaV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and alphaV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating alphaV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.

Figures

Figure 1.

Increased expression of uPA/uPAR and αVβ3 and enhanced Plg binding upon M6P/IGF2R knockdown. (A) Surface and total expression of the indicated molecules in M6P/IGF2R silenced versus control shRNA transduced TCL-598 cells was analyzed by flow cytometry. The values show the percentage of up-regulated mean fluorescence intensity on M6P/IGF2R-silenced cells. Similar results were obtained in three other independent experiments. (B) The mRNA levels of the indicated molecules were analyzed using real-time PCR as described in Materials and Methods. The data represents three independent experiments (C) Cell surface binding of Cy5 labeled human Plg was analyzed by flow cytometry. Cy5-labeled BSA was used as a negative control. The bar graph shows the percentage of up-regulated mean Cy5 fluorescence intensity on M6P/IGF2R-silenced cells (n = 3; p = 0.03).

Figure 2.

Enhanced cell surface uPA/plasmin activity and invasion of M6P/IGF2R knockdown cells. (A) Plasmin activity of M6P/IGF2R-silenced and control shRNA-transduced TCL-598 cells was analyzed by using the plasmin-specific chromogenic substrate S-2251. Plasmin activity was monitored at OD 405 nm by using an ELISA reader. The indicated inhibitors were added to the cells 20 min before adding Plg. (B) uPA activity was measured under the same conditions as in A by using the uPA-specific chromogenic substrate S-2444. (C) Cells were preincubated with the indicated blocking antibodies for 24 h, and plasmin activity was analyzed as described in A. Shown is a representative result of two independent experiments. (D) TCL-598 cells were analyzed in a Boyden chamber cell invasion assay using EGF (10 ng/ml) and FCS as chemoattractants. The concentrations of the used inhibitors: galardin (10 μM), amiloride (10 μM), pepstatin A (10 μM), IAA (10 μM), TA (5 mM), aprotinin (10 μg/ml), and E-64 (10 μM). Results are representative for two independent experiments.

Figure 3.

Enhanced uPA surface expression and plasmin activity in immortalized HUVECs silenced for M6P/IGF2R. HUVECs were immortalized by using telomerase reverse transcriptase (tert) and transduced with either M6P/IGF2R shRNA or control shRNA. (A) Surface and total expression of the indicated molecules was analyzed by flow cytometry. Histograms of a typical result are shown. The values indicate the percentage of altered mean fluorescence intensity of the M6P/IGF2R-silenced cells in comparison with the control cells. (B) Plasmin activity of M6P/IGF2R-silenced and control HUVECtert was analyzed by using the plasmin specific chromogenic substrate S-2251. Plasmin activity was measured at OD 405 nm using an ELISA reader. PAI-1 was used at 10 U/ml. Other inhibitors were used as described in Figure 2. The shown data represent at least two independent experiments.

Figure 4.

Genetic rescue of enhanced plasmin activity and cell invasion in M6P/IGF2R knockdown cells. (A) TCL-598 cells stably transduced with M6P/IGF2R shRNA or the control shRNA were transiently transfected with 1 μg siRNA against uPAR, αV integrin, or both. A scrambled nonspecific siRNA was used as control. Forty-eight hours after transfection, the cells were fixed, permeabilized, and analyzed for expression of the targeted molecules by flow cytometry. The values indicate the percentage of knockdown efficiency measured by the reduction of the mean fluorescence intensity. (B) Forty-eight hours after siRNA transfection, single, double, and triple knockdown cells were analyzed for cell surface plasmin activity as described in Figure 2A. (C) Alternatively the siRNA-transfected cells were seeded on a matrigel coated Boyden chamber, and cell invasion was analyzed as described in Materials and Methods. A representative phase contrast picture of crystal violet stained cells that have invaded through the matrigel is shown. (D) Two fields on three independent Boyden chamber filters of each specimen were evaluated for the number of invading cells. The mean number of cells per field is depicted (n = 2).

Figure 5.

Enhanced cell adhesion and motility in M6P/IGF2R silenced cells. (A) Adhesion of TCL-598 cells on the indicated matrix proteins was determined as described in Materials and Methods. (B) Single and double knockdown cells characterized in Figure 4 were analyzed for their adhesion to vitronectin. (C) Ovarian carcinoma cells (OVMZ-6) transduced with M6P/IGF2R shRNA or control shRNA were analyzed for cell adhesion on the indicated matrix proteins. (D) Migration of OVMZ-6 and TCL-598 cells was tested in a Boyden chamber chemotaxis assay. EGF (10 ng/ml), IGF2 (10 ng/ml), or pro-uPA (10 nM) were used as chemoattractants. Adherent or transmigrated cells were fixed and stained with crystal violet. The stained cells were quantified at OD595 nm using an ELISA reader as described in Materials and Methods. VN, vitronectin; FN, fibronectin; COIV, collagen type IV; COI, collagen type I; BSA, bovine serum albumin.

Figure 6.

Coimmunoprecipitation of M6P/IGF2R, uPAR, and αV integrin. (A) Lysates from TCL-598 kidney carcinoma cells were subjected to immunoprecipitation by using different mAbs as depicted against M6P/IGF2R, uPAR, and various integrin α chains. The precipitates were analyzed by probing the Western blot with the uPAR mAb C8. (B) Some of the precipitates from A were also analyzed by immunoblotting with the polyclonal antibody Ab32815 against M6P/IGF2R.

Figure 7.

Dominance of full-length uPAR on the surface of M6P/IGF2R silenced cells. (A) uPA was precipitated from TCL-598 cell lysates by using the anti-uPA mAb. Coprecipitation of uPA and uPAR was compared in control and M6P/IGF2R knockdown cells by immunoblotting the precipitates with the anti-uPAR mAb C8. (B) Surface biotinylated cells were lysed and subjected to precipitation with streptavidin-coated Sepharose beads. Then, the precipitates were analyzed by immunoblotting with the polyclonal uPAR Ab. (C) The streptavidin precipitates from B were quantified by analyzing the integrated intensity (I.I.) of the uPAR bands on the immunoblot by using the Odyssey infrared imaging system (LI-COR Biosciences). The mean ratio of full-length uPAR to truncated D2D3 was calculated from five independent experiments (p = 0.02; n = 5). (D) Quantification of total surface uPAR (D1D2D3 + D2D3) in control and knockdown cells (n = 5). The total expression of uPAR in the control cells was set as 100% I.I.

Figure 8.

uPAR cleavage but not uPAR internalization and turnover is dependent on M6P/IGF2R expression. (A) TCL-598 cells were stained on ice with directly labeled anti-uPAR mAb H2. After incubation for the indicated times at 37°C, remaining cell surface mAbs were stripped with acid. Cells were analyzed for stripping-resistant (internalized) fluorescence by flow cytometry. Geometric mean values were calculated as percentage of internalized fluorescence (n = 4). (B) Surface-biotinylated TCL-598 cells were incubated in culture medium supplemented with Plg (50 nM) for the indicated time points at 37°C. After incubation, the cells were lysed and biotinylated uPAR was analyzed by SDS-PAGE and immunoblotting. (C) Quantification of experiments shown in B; uPA activity was blocked by adding amiloride (10 μM). The bands were quantified by analyzing the integrated intensity (I.I.) on the immunoblot using the Odyssey infrared imaging system (LI-COR Biosciences). The uPAR values were normalized for beta1 integrin and the percentage of decrease of full-length uPAR (D1D2D3) over time was calculated (n = 6). (D) Surface-biotinylated M6P/IGF2R-negative mouse fibroblasts transduced with human uPAR and human M6P/IGF2R were incubated in culture medium supplemented with human uPA (10 nM) for the indicated times at 37°C. After incubation, the cells were lysed and biotinylated uPAR was analyzed as described in B. (E) Quantification of the integrated intensities of full-length uPAR (D1D2D3) normalized with an unspecific band on the immunoblot. The percentage of decrease of full-length uPAR (D1D2D3) over time was calculated (n = 3).