Thrombospondin-4 mediates TGF-β-induced angiogenesis

Abstract

TGF-β is a multifunctional cytokine affecting many cell types and implicated in tissue remodeling processes. Due to its many functions and cell-specific effects, the consequences of TGF-β signaling are process-and stage-dependent, and it is not uncommon that TGF-β exerts distinct and sometimes opposing effects on a disease progression depending on the stage and on the pathological changes associated with the stage. The mechanisms underlying cell- and process-specific effects of TGF-β are poorly understood. We are describing a novel pathway that mediates induction of angiogenesis in response to TGF-β1. We found that in endothelial cells (EC) thrombospondin-4 (TSP-4), a secreted extracellular matrix (ECM) protein, is upregulated in response to TGF-β1 and mediates the effects of TGF-β1 on angiogenesis. Upregulation of TSP-4 does not require the synthesis of new protein, is not caused by decreased secretion of TSP-4, and is mediated by activation of SMAD3. Using Thbs4^-/- mice and TSP-4 shRNA, we found that TSP-4 mediated pro-angiogenic functions in cultured EC and angiogenesis in vivo in response to TGF-β1. We observed~3-fold increases in tumor mass and levels of angiogenesis markers in animals injected with TGF-β1, and these effects did not occur in Thbs4^-/- animals. Injections of an inhibitor of TGF-β1 signaling SB-431542 also decreased the weights of tumors and cancer angiogenesis. Our results from in vivo angiogenesis models and cultured EC document that TSP-4 mediates upregulation of angiogenesis by TGF-β1. Upregulation of pro-angiogenic TSP-4 and selective effects of TSP-4 on EC may contribute to stimulation of tumor growth by TGF-β despite the inhibition of cancer cell proliferation.

Figure 1. Treatment of microvascular EC with TGF-β1 increases TSP-4 levels

A: RF/6A cells and HDMEC were stimulated with 10 ng/ml TGF-β1 for 24 h. TSP-4, TSP-1, TSP-3, and β-actin were visualized with corresponding antibodies in Western blotting as described in the Methods. B: MLEC and RF/6A cells were treated with 10 ng/ml TGF-β1 or bFGF; TSP-4 and β-actin were detected in Western blotting. C: Human VSMC were treated with TGF-β1 for indicated time periods; TSP-4, total SMAD3, phosphorylated SMAD3, and β-actin were detected in Western blots.

Figure 2. TGF-β1 regulates TSP-4 at the level of protein stability

A: TSP-4 mRNA in cells stimulated with TGF-β1. RF/6A microvascular EC were stimulated with 10 ng/ml of TGF-β1 for 6 hours, and TSP-4 mRNA levels were analyzed by Quantitative RT-PCR. B: Promoter-reporter constructs were transiently transfected to RF/6A cells as described in Methods, and cells were stimulated with TGF-β1 the next day for 24 hours. Luciferase activity was measured in cell lysates. C: Cell culture supernatants (60 μl) form RF/6A stimulated with TGF-β1 were analyzed by Western blotting with anti-TSP-4 antibody. SIS3 = cells pre-treated with SIS3 for 30 min as described in Methods; SB and SB431542 = cells pre-treated with SIS3 for 30 min as described in Methods. D: RF/6A cells were pre-treated with 10 μg/ml cyclohexamide for 30 min and stimulated with TGF-β1 and analyzed by Western blotting with anti-TSP-4.

Figure 3. SMAD3 mediates upregulation of TSP-4 in response to TGF-β1

A, B: RF/6A and MLEC were transfected with lentiviral particles expressing SMAD3 shRNA (see Methods). Cells were stimulated with TGF-β1 for 24 h and lysed, and proteins were separated in SDS-PAGE followed by Western blotting. TSP-4, β-actin, and SMAD3 were detected using specific antibodies. C: RF/6A cells were pre-treated with the inhibitor of SMAD3 SIS3 as described in the Methods and stimulated with TGF-β1 and analyzed by Western blotting with anti-TSP-4.

Figure 4. TSP-4 mediates increased adhesion and migration of microvascular EC in response to TGF-β1

A: TGF-β1-stimulated MLEC from WT and Thbs4^−/− (KO) mice were seeded into 24-well plates coated with fibronectin and incubated at 37 °C for 1 h. Unattached cells were removed by washing, and the remaining DNA in the wells was measured using the Cyquant reagent. Adhesion of TGF-β1-stimulated MLEC was compared to the adhesion of non-stimulated cells. *p<0.05 compared to non-stimulated cells, # p<0.05 compared to cells from WT mice; n=3. B: Experiments identical to the ones described in panel A were performed with cultured MAEC; n=3. C: Migration of TGF-β1-stimulated MLEC from WT and Thbs4^−/− mice was measured in non-coated Boyden chambers with 8 μm pores and compared to migration of non-stimulated MLEC. DNA in the lower chamber was measured after 4 h at 37 °C. The values are expressed as percent of average migration of non-stimulated cells. *p < 0.05 compared to control (non-stimulated cells), # p<0.05 compared to cells from WT mice; n=3. D: Experiments identical to the ones described in A were performed in MAEC from WT and Thbs4^−/− mice. E: Proliferation of TGF-β1-stimulated EC: 12-well plates were coated with fibronectin overnight at 4 °C. EC from WT mice were added and allowed to proliferate at 37 °C for 24, 48, 72, and 96 h; n=3. The amount of DNA in the wells was measured at 24, 48, 72, and 96 h after seeding the cells. The average values of fluorescence are presented as % of average values of fluorescence in control wells with non-stimulated cells.

Figure 5. TSP-4 mediates increased angiogenesis in response to TGF-β1 in Matrigel plug angiogenesis model

20 ng/ml TGF-β1 was added to Matrigel, and Matrigel was injected subcutaneously to WT and Thbs4^−/− (KO) mice. A: Quantification of anti-CD31 staining (EC) in Matrigel plugs in WT and Thbs4^−/− mice (n=10). B: Quantification of anti-α-actin staining (SMC) in Matrigel plugs in WT and Thbs4^−/− mice (n=10). The stained area was quantified using ImagePro6.1. *p < 0.05 compared to control (non-stimulated cells), # p<0.05 compared to cells from WT mice. C: Quantification of TSP-4 (n=10), *p < 0.05.

Figure 6. Effect of TSP-4 and SMAD3 shRNA on angiogenesis in response to TGF-β1 in Matrigel plug angiogenesis model

Matrigel supplemented with TSP-4-, SMAD3-, or control-shRNA-expressing lentiviral particles was injected subcutaneously to WT mice as described in the Methods. TGF-β1 was administered in daily IP injections. A: Quantification of anti-vWF (left panel), anti-α-actin (middle panel), and anti-TSP-4 (right panel) staining in Matrigel plugs of mice injected with Matrigel containing TSP-4 shRNA or control shRNA (n=10). B: Quantification of anti-vWF (left panel), anti-α-actin (middle panel), and anti-TSP-4 (right panel) staining in Matrigel plugs of mice injected with Matrigel containing SMAD3 shRNA or control shRNA (n=10). The stained area was quantified using ImagePro6.1. *p < 0.05 compared to control shRNA, # p<0.05 compared to control (injected with PBS) mice.

Figure 7. TSP-4 mediates angiogenesis in response to TGF-β1 in a cancer model

A: Mouse breast cancer EMT6 cells were injected into mammary fat pad of WT or Thbs4^−/− (KO) mice. Mice received daily IP injections of TGF-β1. A: representative tumors from one of the experiments. B: tumor weight, n=10, *p < 0.05 compared to control mice injected with PBS; # p<0.05 compared to WT mice. C: Angiogenesis marker CD31 (EC) was visualized by immunohistochemistry in frozen sections of tumors. D: α-actin (SMC) was visualized by immunohistochemistry in frozen sections of tumors from WT and Thbs4^−/− mice. E: TSP-4 was visualized by immunohistochemistry in frozen sections of tumors from WT and Thbs4^−/− mice. A–D: Mean stained area, % × mean tumor weight, n = 10, *p < 0.05 compared to WT, # p<0.05 compared to WT mice.

Figure 8. Effect of inhibitor of TGF-β receptors SB-431542 on cancer growth and angiogenesis

In the experiment similar to those described in Fig. 7, 4.2 mg/kg SB-431542 in 0.1 ml of PBS or PBS alone was injected IP daily 30 min before the injection of TGF-β1. A: EMT6 tumor weight and representative tumors, n=10, ***p<0.005, *****p<0.00005. B: Levels of CD31 in tumors, IHC, n=10. C: Levels of NG2 in tumors, IHC, n=10. D: Levels of phospho-SMAD3 in tumors, IHC, n=10. E: Levels of TSP-4 in tumors, IHC, n=10.