Suppression of uPA and uPAR attenuates angiogenin mediated angiogenesis in endothelial and glioblastoma cell lines

Abstract

Background: In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.

Methodology/principal findings: In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.

Conclusions/significance: Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.

Figure 1. shRNA against uPA and uPAR inhibit tumor-induced angiogenesis.

(A–B) U87 and U87 SPARC-overexpressing cells were transfected as described in Materials and Methods. Briefly, U87, U87 SPARC-overexpressing cells and HMEC seeded at a density of 1.5×10⁵ cells/100 mm plate were serum-starved for 4–6 hrs, transfected with 8 µg of SV (Scrambled vector) puPA, puPAR or pU2 for 12 hrs, and serum-containing medium was added. After 48 hrs, 3 mL of serum-free medium were added overnight and tumor-conditioned medium was collected and fibrin zymography analysis was done to detect uPA activity. The band intensities of uPA activity were measured and normalized with the intensity of mock-conditioned medium band. uPA activity levels were measured in (A) U87 MG and (B) U87 SPARC-overexpressing cells and (C) HMEC cells by fibrin zymography and uPA/uPAR levels were determined by Western blotting. (D) In vitro angiogenesis assay. The tumor-conditioned medium was added into 48-well plates, which were coated with Matrigel and pre-seeded with HMEC (2×10⁴ cells per well). After overnight incubation at 37°C, cells were observed under the bright field microscope for the formation of capillary-like structures. (E) In vivo angiogenic assay using the dorsal skin-fold model. Briefly, the animals were implanted with diffusion chambers containing Mock, pSV, puPA, puPAR and pU2-transfected cells in a dorsal cavity. Ten days after implantation, the animals were sacrificed and the skin-fold covering the diffusion chamber was observed under a bright field microscope for the presence of tumor-induced neovasculature (TN) and pre-existing vasculature (PV) and photographed.

Figure 2. shRNA-mediated downregulation of uPAR and uPA increases HMEC apoptosis and reduces U87, U87 SPARC and HMEC proliferation.

(A) U87, (C) U87 SPARC and (E) HMEC were transfected with pSV, puPA, puPAR or pU2, and the cells were trypsinized followed by PI staining as per standard protocols. 10,000 cells were sorted by flow cytometer to determine the DNA content of the cells. (B), (D) and (F) Briefly, 1.5×10⁵ U87, U87 SPARC and HMEC were transfected with pSV, puPAR, puPA or pU2 in vitronectin-coated 96-well microplates under serum-free conditions. The number of viable cells was assessed by MTT assay. *p<0.05, **p<0.01. (G) Graph represents the percentage of apoptosis in cells before and after transfection with shRNA. Data shown are the mean ± SD values from four different experiments. **p<0.01.

Figure 3. shRNA against uPA and uPAR downregulates secreted pro-angiogenic factors.

HMEC and cancer cell co-cultures were incubated with Ray Biotech angiogenesis antibody array 1 and 2 as per the manufacturer's instructions (Fig. A & C). Results are represented from three independent experiments. Highlighted proteins are angiogenin, epidermal growth factor (EGF), GRO, IL-6 (interleukin 6), GM CSF (granulocyte macrophage colony stimulating factor), and Ang-1 (angiopoeitin-1). (B) and (D) Graphs represent quantification of antibody array using NIH ImageJ software. *p<0.05, **p<0.01.

Figure 4. shRNA against uPA and uPAR inhibits secreted levels of ANG, Ang-1 and VEGF in cancer cells, endothelial cells and co-cultures.

(A–C) 1.5×10⁵ U87 and U87 SPARC cells were transfected as described earlier. Conditioned medium was collected after 72 hrs and assayed for (A) ANG, (B) Ang-1, and (C) VEGF levels by ELISA. Data represented are the average of three independent experiments. *p<0.01, **p<0.001. (D–F) HMEC cells, U87 co-cultures and U87 SPARC co-cultures were transfected with jet Prime shRNA transfection reagent as mentioned in the Materials and Methods. The conditioned medium was collected and assayed for ANG, Ang-1 and VEGF by ELISA. Data represented are the average of three independent experiments. (G–I) HMEC cells transfected with shRNA constructs were lysed with RIPA lysis buffer and immunoblotted with primary antibodies for ANG, Ang-1 and VEGF and corresponding secondary antibodies. A representative blot of three independent experiments is shown. The blots were probed for GAPDH to show equal loading of lysates.

Figure 5. shRNA against uPA and uPAR inhibits nuclear localization of ANG and 45S rRNA gene expression and decreases ribonucleolytic activity in HMEC cells.

(A) Serum-starved endothelial cells (30 to 40% confluence) left untreated, treated with exogenous ANG (250 ng/mL for 48 hrs), pretreated with neomycin (100 µM for 1 hr), or treated with rANG and then with neomycin were taken and nuclear extracts were prepared and western blotted for ANG. The purity of the nuclear extracts was tested by probing for Lamin B (middle panel) and tubulin (cytoplasmic marker) (bottom panel). (B) Localization of ANG in nuclei of endothelial cells. Serum-starved (30 to 40%) cells were left untreated or transfected with pU2 for 48 hrs and stained for ANG (green) after permeabilization. Arrows indicate angiogenin. (C) puPA, puPAR and pU2 inhibit the ribonucleolytic activity of HMEC. Ribonucleolytic activity of ANG isolated from puPA-, puPAR- and pU2-transfected cells was done as described by Shapiro et al. *p<0.01, **p<0.001. (D) Fold induction of 45S rRNA gene expression in mock and empty vector-transfected cells was quantified using real-time RT-PCR. (E) HMEC cells (30 to 40% confluence) were left untreated, transfected with pEV, puPA, puPAR or pU2, and real-time RT-PCR was performed for 45S rRNA gene expression. The results shown in panel E represent the averages of standard deviation of data from three independent experiments that were normalized to the β actin expression levels. The 45S rRNA levels were normalized to uninfected cells and were assigned a value of “1” for comparison. *p<0.05, **p<0.001.

Figure 6. Exogenous addition of rAng activates uPA and ruPA increases angiogenin levels in endothelial cells.

(A) Serum-starved HMEC cells were left untreated or pretreated with 500 ng of rANG. Conditioned medium was collected at 3, 6, 24 and 48 hrs time points. uPA levels were then determined by fibrin zymography. The gel shown is a representative of three independent experiments. (B) Serum-starved endothelial cells either left untreated or pretreated with 500 ng ruPA were subjected to quantitative estimation of ANG by ELISA at different time points. The data represent the average concentration of angiogenin in pg/mL of three independent experiments (p<0.05). (C) Serum-starved endothelial cells were left untreated, treated with different concentrations of ruPA, or treated with ruPA followed by different concentrations of ruPA ATF for 24 or 48 hrs. Lysates were collected and checked for the presence of angiogenin by western blotting. GAPDH was probed to verify equal loading. (D) Serum-starved endothelial cells were left untreated or treated with 500 ng ruPA, treated with 500 ng ruPA ATF for 48 hrs, and conditioned medium was collected. Angiogenin levels were quantified by ELISA. Results shown here are from three separate independent experiments (**p<0.01).

Figure 7. shRNA against uPA and uPAR downregulates expression of Tie-2, GRB2 and phosphorylation of AKT, ERK and BAD in HMEC cells.

(A) Western blot analysis of shRNA-transfected HMEC was carried out with primary antibodies against Tie-2, GRB2, and phosphorylated forms of AKT, T-AKT, ERK, BAD and total BAD. GAPDH antibody was also immunoblotted to demonstrate equal loading of lysates. (B) Nuclear extracts were isolated from shRNA-transfected endothelial cells and probed for phospho Forkhead in Rhabdosarcoma (FKHR) and total FKHR by western blotting. The purity of the nuclear extracts was checked by blotting for Lamin B (nuclear) and alpha tubulin (cytoplasmic). (C) Forkhead in Rhabdosarcoma (FKHR transcription factor activation assay in HMEC after transfection with shRNA against uPA and uPAR alone and in combination. Nuclear extracts prepared from untreated, pSV-, puPA-, puPAR- and pU2-transfected shRNA treated cells were checked for FKHR activation by TransAM FKHR transcription factor assay as per the manufacturer's instructions. The assay was performed in triplicate, and results were similar.

Figure 8. Schematic diagram showing the possible mechanisms by which shRNA against uPA and uPAR inhibits angiogenesis in glioblastoma cell lines.

(A) uPA upon binding with uPAR activates secretion of angiogenin. Angiogenin secreted by cancer cells and endothelial cells binds to a unknown endothelial receptor, forming an actin angiogenin complex and is translocated to the nucleus by a microtubule lysosomal independent pathway. Angiogenin is then transported to the nucleus where it interacts with fibrillarin in semi-confluent endothelial cells and activates 45S rRNA ribosome gene synthesis. uPA interaction with uPAR also activates Ang-1 in endothelial cells and this secreted Ang-1 by endothelial cells phosphorylates Tie-2 receptor and activates FKHR in the GRB2/Akt/BAD pathway. In panel B, in shRNA-transfected cells, as uPA and uPAR interaction is negligible, ANG secreted by endothelial cells is not translocated to the nucleus, 45S rRNA synthesis is inhibited, and ribonucleolytic activity of ANG is significantly retarded. Ang-1 induced Tie-2 phosphorylation, Forkhead in Rhabdosarcoma (FKHR) activation and the GRB2/AKT/ERK/BAD pathway was significantly retarded in shRNA-transfected endothelial cells.