Distinct angiogenic mediators are required for basic fibroblast growth factor- and vascular endothelial growth factor-induced angiogenesis: the role of cytoplasmic tyrosine kinase c-Abl in tumor angiogenesis - PubMed (original) (raw)

Distinct angiogenic mediators are required for basic fibroblast growth factor- and vascular endothelial growth factor-induced angiogenesis: the role of cytoplasmic tyrosine kinase c-Abl in tumor angiogenesis

Wei Yan et al. Mol Biol Cell. 2008 May.

Abstract

Signaling pathways engaged by angiogenic factors bFGF and VEGF in tumor angiogenesis are not fully understood. The current study identifies cytoplasmic tyrosine kinase c-Abl as a key factor differentially mediating bFGF- and VEGF-induced angiogenesis in microvascular endothelial cells. STI571, a c-Abl kinase inhibitor, only inhibited bFGF- but not VEGF-induced angiogenesis. bFGF induced membrane receptor cooperation between integrin beta(3) and FGF receptor, and triggered a downstream cascade including FAK, c-Abl, and MAPK. This signaling pathway is different from one utilized by VEGF that includes integrin beta(5), VEGF receptor-2, Src, FAK, and MAPK. Ectopic expression of wild-type c-Abl sensitized angiogenic response to bFGF, but kinase dead mutant c-Abl abolished this activity. Furthermore, the wild-type c-Abl enhanced angiogenesis in both Matrigel implantation and tumor xenograft models. These data provide novel insights into c-Abl's differential functions in mediating bFGF- and VEGF-induced angiogenesis.

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Figures

Figure 1.

STI571 inhibits HMVEC proliferation induced by bFGF and VEGF. HMVECs were pretreated with bFGF (5 ng/ml) or VEGF (10 ng/ml) in the absence or presence of STI571 (10 μM) for 24 h. [3H]thymidine (1 μCi) was added for 6 h, and radioactivity was quantified. *p < 0.05 compared with control cells; +p < 0.05 compared with the cells treated with bFGF or VEGF. n = 6.

Figure 2.

Divergent effects of STI571 on bFGF- and VEGF-induced angiogenesis. (A) STI571 inhibits bFGF- but not VEGF-induced cell migration. HMVECs were loaded into the upper chamber of transwells, and bFGF (5 ng/ml) or VEGF (10 ng/ml) was added to the low chamber in the absence or presence of STI571 (10 μM) for 4 h. Cells migrated to the membrane were quantitated. n = 5. (B) STI571 blocks bFGF- but not VEGF-induced tube formation. The cells were loaded onto the Matrigel and incubated for 12 h in the presence of bFGF (5 ng/ml), VEGF (10 ng/ml), or STI571 (10 μM). The tubes density was quantitated. n = 4. (C) Dose-dependent effects of bFGF and VEGF on tube formation. The indicated different doses of bFGF and VEGF in the absence or presence of STI571 were added to the cells, and the extent of tube formation was quantitatively analyzed. n = 4. (D) STI571 blocks bFGF- but not VEGF-induced cell survival activity. The cells were treated with serum-free medium for 5 d in the presence of bFGF (5 ng/ml), VEGF (10 ng/ml), or STI571 (10 μM). The survival cells were quantified. n = 3. (E) STI571 inhibits cell migration induced by bFGF but not VEGF in HMEC-1. The cell migration and tube formation were measured similarly as in A, except HMEC-1 instead of HMVEC. n = 4. *p < 0.05 compared with control; +p < 0.05 compared with bFGF-treated cells.

Figure 3.

bFGF induces c-Abl activation and protein expression. (A) After treatment with serum-free medium for 12 h, the cells were stimulated with bFGF (5 ng/ml) for 2, 5, and 10 min in the absence or presence of STI571 (10 μM). The cell lysates were subjected for immunoblotting using antibodies against c-Abl pY245, total c-Abl, and actin. c-Abl pY245 level was evaluated by densitometry analyses followed by normalizing with actin. (B) The cells were treated with bFGF or VEGF (5 ng/ml) for 12 h. The cell lysates were used for immunoblotting using anti-c-Abl antibody. c-Abl level was evaluated by normalizing with actin. *p < 0.05 compared with control at the zero time; +p < 0.05 compared with corresponding time cells treated with bFGF alone. n = 3–4.

Figure 4.

c-Abl mediates bFGF-induced signaling cascade, whereas Src mediates VEGF-induced signaling activation. (A) bFGF induces association of integrin β3 with FGFR, and VEGF induces association of integrin β5 with Flk-1/KDR. Cells were stimulated with bFGF (5 ng/ml) or VEGF (10 ng/ml) for 5 min, and cell lysates were immunoprecipitated with anti-integrin β3, β5, Flk-1/KDR, or FGFR antibody, followed by immunoblotting using anti-FGFR, Flk-1/KDR, pY20, Src, Src pY418, β3, or β5 antibody. C, F, and V indicate control, bFGF, and VEGF, respectively. (B) bFGF stimulates FAK activation. The cells were stimulated with bFGF (5 ng/ml) for 5 and 15 min and then subjected to immunoblotting using anti-FAK pY397 and pY861 antibody. (C) VEGF but not bFGF induces association of FAK pY861 with Src. The cells were stimulated with bFGF (5 ng/ml) or VEGF (10 ng/ml) for 5 min, and cell lysates were immunoprecipitated with anti-Src antibody followed by immunoblotting using anti-FAK and FAK pY861 antibody. (D) bFGF promotes association of c-Abl with FAK pY861 but dissociation from Src. Five minutes after treatment with bFGF (5 ng/ml) or VEGF (10 ng/ml) in the absence or presence of STI571 (10 μM), the cells were harvested, and cell lysates were immunoprecipitated with anti-c-Abl antibody followed by immunoblotting using anti-c-Abl, FAK, FAK pY861, Src, or Src pY418 antibody. (E) STI571 only inhibits bFGF-induced Erk 1 and Erk 2 activation. Cells were stimulated with bFGF (5 ng/ml) or VEGF (10 ng/ml) in the absence or presence of STI571 (10 μM) for 5 min before cell lysis for immunoblotting against Erk 1, Erk 2, pErk 1, or pErk 2. The phosphorylated Erk 1 and Erk 2 were quantitated by normalizing with nonphosphorylated Erk 1 and Erk 2. *p < 0.05 compared with control. n = 3.

Figure 5.

STI571 blocks bFGF-induced cell cytoskeleton reorganization. (A) Cells were treated with bFGF (5 ng/ml) or VEGF (10 ng/ml) for 5 and 30 min. Then, the cells were immunostained using rhodamine phalloidin. (B) The cells were stimulated with bFGF (5 ng/ml) or VEGF (10 ng/ml) in the absence or presence of STI571 (10 μM) for 5 min followed by cytoskeleton staining. Arrows indicate the ruffling of plasma membrane. The images show one representative of three individual experiments. Bars, 10 μm.

Figure 6.

Overexpression of WT and MT c-Abl results in altered protein interaction and activation. (A) Ectopic expression of WT c-Abl increases both active c-Abl level and its association with FAK pY861. Cell lysates from cells expressing vector control, WT, or MT c-Abl were used for immunoblotting to determine c-Abl expression or immunoprecipitation with anti-pY20 or c-Abl antibody before immunoblotting using anti-c-Abl, FAK, or FAK pY861 antibody. C, parental cells as control; V, cells engineered with an empty retroviral vector. (B) Cells expressing WT c-Abl exhibit changes in protein interaction. Cells ectopically expressing different versions of c-Abl were harvested, and cell lysates were used for immunoprecipitation using anti-integrin β3 or β5 antibody, followed by immunoblotting using antibodies against FGFR, Flk-1/KDR, or Src. (C) WT c-Abl enhances Erk 1 and Erk 2 activation in response to bFGF, but MT c-Abl blocks this activity. Cells ectopically expressing different versions of c-Abl were stimulated with bFGF (5 ng/ml) or VEGF (10 ng/ml) for 5 min, and then the lysates were used for immunoblotting to examine phosphorylated or nonphosphorylated Erk 1 and Erk 2. The phosphorylated Erk 1 and Erk 2 were quantitated by normalizing with nonphosphorylated Erk 1 and Erk 2. *p < 0.05 compared with respective control in each group; +p < 0.05 compared with the levels in either parental or vector control group treated with bFGF. n = 3.

Figure 7.

Divergent effects of WT and MT c-Abl on angiogenesis in vitro. After treatment with bFGF (5 ng/ml) or VEGF (10 ng/ml), cells expressing different versions of c-Abl were measured for cell proliferation and tube formation (A). *p < 0.05 compared with respective control in each group; +,#p < 0.05 compared with cells treated with respective growth factor in the parental and vector control groups. After treatment with bFGF or VEGF combined with PD98059 (10 μM), the cells were examined for cell migration (B). *p < 0.05 compared with respective control in each group, +p < 0.05 compared with the cells treated with growth factors in the absence of PD98059; #p < 0.05 compared with the cells treated with bFGF in the parental and vector control groups. n = 4–6.

Figure 8.

Distinct effects of c-Abl on angiogenesis in vivo. (A) STI571 inhibits angiogenesis induced by bFGF in Matrigel implantation model. HMVECs premixed with Matrigel in the presence of bFGF or VEGF (1 μg) were subcutaneously injected into mice (three mice in each group). After the mice were treated with vehicle alone as control or STI571 for 10 d, Matrigel plugs were extracted for the analysis of CD31 staining as described in Materials and Methods. An insert represents a Matrigel plug. Blood vessel areas were quantified with an image-scanning program developed by the National Institutes of Health. *p < 0.05 compared with the control group; +p < 0.05 compared with the bFGF-treated group. (B) WT and MT c-Abl exhibit opposite effects on bFGF-induced angiogenesis. HMVECs ectopically expressing vector control, WT, or MT c-Abl were mixed with Matrigel in the presence of bFGF or VEGF as above. On day 10, the Matrigel plugs were excised for immunohistochemistry staining of CD31. *p < 0.05 compared with respective untreated control group. (C and D) WT and MT c-Abl exert opposite effects on tumor growth and angiogenesis in xenograft animal model. MDA-MB-231 cells and HMVECs expressing control or different versions of c-Abl were coinjected into immunodeficient mice as described in Materials and Methods. Tumor sizes were measured weekly for 12 wk (C, n = 6). Removed tumor tissues were stained with an anti-CD31 antibody, and a representative tumor section for each condition is shown. CD31-positive endothelial cells with brown staining indicated vasculature network, and vessel area was analyzed by quantifying CD31-positive staining. *p < 0.05 compared with the control group (D, n = 6).

Figure 9.

A hypothetical model for the signaling pathways induced by bFGF and VEGF. Once endothelial cells are stimulated with bFGF, the cooperation of membrane-anchored receptors between integrin αvβ3 and FGFR leads to the “out-side in” signaling activation. c-Abl disassociates from Src that is connected to integrin β3 and increases in association with activated FAK, resulting in downstream MAPK activation. In addition, c-Abl may directly regulate reorganization of cytoskeleton actin to facilitate cell motility. VEGF engages a similar but c-Abl–independent angiogenic machinery in which Src acts as an upstream effector of FAK.

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Distinct angiogenic mediators are required for basic fibroblast growth factor- and vascular endothelial growth factor-induced angiogenesis: the role of cytoplasmic tyrosine kinase c-Abl in tumor angiogenesis - PubMed (original) (raw)