VEGF Production Is Regulated by the AKT/ERK1/2 Signaling Pathway and Controls the Proliferation of Toxoplasma gondii in ARPE-19 Cells

Abstract

The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.

Keywords: PI3K/MAPK signaling pathways; Toxoplasma gondii proliferation; ocular toxoplasmosis; retinal pigment epithelium; vascular endothelial growth factor.

Figure 1

VEGF production in T. gondii-infected ARPE-19 cells. (A,C) ARPE-19 cells were infected with T. gondii tachyzoites for 24 h, fixed and probed against α-Tubulin. The cells were counterstained with DAPI and visualized by confocal microscope. (B,E) ARPE-19 cells were infected with T. gondii at multiplicity of infection (MOI) of 1, 5, and 10 for 24 h and VEGF mRNA levels were evaluated using qRT-PCR (B) and western blot (E). (D) ARPE-19 cells were infected with T. gondii at MOI 10 for 0.5, 1, 18, and 24 h and the VEGF mRNA levels evaluated using qRT-PCR. Each value represents the mean ± standard deviation (SD). **P < 0.01, ***P < 0.001 compared with control ARPE-19 cells (n = 3). (F–H) ARPE-19 cells were infected with T. gondii at various MOIs of 1 (F), 5 (G), or 10 (H) for 0.5, 1, 18 and 24 h and the VEGF protein levels evaluated using western blot.

Figure 2

VEGF production in the eyes of T. gondii-infected mice. Five mice were sacrificed for the determination of VEGF expression in the eyeball by qRT-PCR (A) and western blot (B). ***P < 0.001 compared with uninfected normal control mice (n = 3).

Figure 3

HIF-1α and HO-1 expressions in T. gondii-infected ARPE-19 cells. (A,B) ARPE-19 cells were infected with T. gondii for 24 h (A) or MOI 10 (B), and protein levels were evaluated using western blot. (C) Nuclear and cytosolic fractions were prepared and the expression level of HIF-1α was evaluated using western blot. Anti-α-Tubulin and anti-fibrillarin were used as the cytosol and nuclear loading control, respectively. Similar results were obtained in three independent experiments (n = 3).

Figure 4

Roles of PI3K/AKT signaling pathways on T. gondii-induced VEGF expression in ARPE-19 cells. (A) ARPE-19 cells were infected with T. gondii at MOI 1, 2, 3, 5, and 10 for 24 h and the phosphorylation of AKT at Ser 473 was analyzed using western blot. (B–E) ARPE-19 cells were preincubated with the PI3K inhibitors LY294002 (B,D), GDC-0941 (C,E) for 1 h followed by infection with T. gondii at MOI 10 for a further 23 h and the VEGF levels were evaluated by western blot and qRT-PCR (B,C) and ELISA (E), respectively. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control or T. gondii-infected cells (n = 3).

Figure 5

Roles of MAPK signaling pathways in T. gondii-induced VEGF expression in ARPE-19 cells. (A) ARPE-19 cells were infected with T. gondii at MOI 10 for 24 h and the activation of MAPK subsets were evaluated at the indicated time points. (B–D) ARPE-19 cells were preincubated with the PD098059 (B), SB203580 (C) and SP600125 (D) for 1 h and infected with T. gondii MOI 10 for a further 23 h. The VEGF mRNA, protein levels and secretion levels were then evaluated by qRT-PCR, western blot and ELISA respectively. Anti-α-Tubulin was used as the internal control. Similar results were obtained in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control or T. gondii-infected cells (n = 3).

Figure 6

Roles of VEGF in activation of PI3K/MAPK signaling pathways in T. gondii-infected ARPE-19 cells. (A,B) ARPE-19 cells were preincubated with anti-VEGF agent bevacizumab (BCM) for 24 or 48 h and then infected with T. gondii at MOI 10 for 24 h. Cells were lysed and the PI3K/MAPK phosphorylation levels were assessed using western blot analysis (A) and quantified (B). Bar plot depicting the p-pAKT/α-Tubulin, p-ERK1/2/α-Tubulin, p-p38/α-Tubulin, and p-JNK/α-Tubulin ratios as determined by densitometric analysis of western blot and expressed as fold change compared with each indicated control group. For all panels, data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with each indicated control group. (C,D) ARPE-19 cells were transfected with VEGF-specific siRNA for 48 h. ARPE-19 cells were preincubated with the GDC-0941, PD098059, SB203580, and SP600125 for 1 h and infected with T. gondii MOI 10 for a further 23 h. (C) Cells were lysed and the PI3K/MAPK phosphorylation levels were assessed using western blot analysis. Anti-α-Tubulin was used as the internal control. (D) The VEGF secretion levels were evaluated by ELISA. Similar results were obtained in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control or T. gondii-infected cells (n = 3).

Figure 7

Effect of anti-VEGF agent bevacizumab on T. gondii invasion and proliferation in ARPE-19 cells. (A,B) ARPE-19 cells were treated with various doses of the bevacizumab (BCM) for 24 h and infected with T. gondii for 2 h (A) or 24 h (B). The cells were stained with Texas Red-X phalloidin (red) and DAPI (blue) to identify F-actin and nuclei, respectively. (C) The number of T. gondii-infected cells and the total number of cells were counted under a fluorescence microscope. (D) ARPE-19 cells were pretreated with various doses of BCM for 24 h, and infected with T. gondii MOI 5 for 2 h, and then uninfected and free tachyzoites were washed off. Cells were incubated for a further 24 h and then fixed and intracellular parasite numbers were evaluated. *P < 0.05, **P < 0.01, ***P < 0.001 compared with T. gondii-infected control ARPE-19 cells (n = 3).

Figure 8

Effects of VEGF in T. gondii proliferation and TP3 expression. (A) ARPE-19 cells were transfected with VEGF-specific siRNA for 48 h and subsequently infected with T. gondii for 24 h. (B) Cells were fixed and stained with Texas Red-X phalloidin and DAPI. Intracellular GRP-RH parasites were revealed by fluorescence microscope and counted GFP parasites in 100 cells. All data shown are representative of three independent experiments. (C) VEGF knockdown efficiency was determined by qRT-PCR. (D) VEGF and T. gondii TP3 protein levels were detected by western blot. α-Tubulin was used as loading control. Data are representative of three independent experiments. **P < 0.01, ***P < 0.001 compared with control, control siRNA and/or T. gondii-infected cells (n = 3).

Figure 9

Effect of VEGF-R2 antagonist SU5416 on the regulation of VEGF production and parasite proliferation in T. gondii-infected ARPE-19 cells. ARPE-19 cells were pre-incubated with the indicated concentration of SU5416 for 1 h and infected with T. gondii MOI 10 for a further 23 h. (A) The VEGF secretion levels were evaluated by ELISA. (B) Cells were stained with Texas Red-X phalloidin and DAPI. Intracellular GFP-RH tachyzoites were revealed by fluorescence microscope and counted GFP parasites in 100 cells. ARPE-19 cells were preincubated with the DMSO, SU5416, GDC-0941, and PD098059 for 1 h and infected with T. gondii MOI 10 for a further 23 h. (C) The expression of listed proteins were determined by using western blot analysis. Anti-α-Tubulin was used as the internal control. (D) The VEGF secretion levels were evaluated by ELISA. Similar results were obtained in three independent experiments. **P < 0.01, ***P < 0.001 compared with indicated control cells (n = 3).

Figure 10

Scheme of functional relationship between T. gondii, VEGF and PI3K/MAPK signaling pathways in the parasite infected ARPE-19 cell.