Slit2N and Robo4 regulate lymphangiogenesis through the VEGF-C/VEGFR-3 pathway

Abstract

Background: Signaling through vascular endothelial growth factor C (VEGF–C) and VEGF receptor 3 (VEGFR-3) plays a central role in lymphangiogenesis and the metastasis of several cancers via the lymphatics. Recently, the Slit2/Robo4 pathway has been recognized as a modulator of vascular permeability and integrity. Signaling via the Robo receptor inhibits VEGF-mediated effects; however, its effects on lymphatic endothelial cell function have not been well characterized.

Results: We found that pretreatment with Slit2N, an active fragment of Slit2, inhibited VEGF-C-mediated lung-derived lymphatic endothelial cell (L-LEC) proliferation, migration, and in vitro tube formation. Slit2N induced the internalization of VEGFR-3, which blocked its activation, and inhibited the activation of the PI3K/Akt pathway by VEGF-C in L-LECs. Moreover, we found that inhibition of VEGF-C-induced effects by Slit2N was Robo4-dependent.

Conclusion: These results indicate that Slit2N/Robo4 modulates several key cellular functions, which contribute to lymphangiogenesis, and identify this ligand-receptor pair as a potential therapeutic target to inhibit lymphatic metastasis of VEGF-C-overexpressing cancers and manage lymphatic dysfunctions characterized by VEGF-C/VEGFR-3 activation.

Figure 1

Slit2N inhibits VEGF-C-enhanced growth, migration and tube formation of L-LECs. (A) Proliferation of L-LECs as assessed by MTS assay after incubation with control, 10 nM Slit2N, or VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05). (B) Transwell migration of L-LECs after treatment with control, 10 nM Slit2N, or VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (***p < 0.001). (C) Representative tube formation assay on ECM of L-LECs after treatment with control, 10 nM Slit2N, or VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C. (D) Average length of L-LEC tubes as assessed by in vitro tube formation assay on ECM after treatment with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C, relative to average tube length of untreated cells (Slit2N “-”, VEGF-C “-”). Data represent the mean ± SD of 3 independent experiments (*p < 0.05). (E) Representative Western blot analysis of Slit2N expression in L-LECs transduced with a control adenovirus (Ctrl) or with an adenovirus expressing V5-tagged Slit2N. GAPDH used as loading control. (F) Average length of L-LEC tubes in L-LECs transduced with a control adenovirus (Slit2N-Adenovirus “-”) or in L-LECs transduced with a Slit2N-expressing adenovirus (Slit2N-Adenovirus “+”) as assessed by in vitro tube formation assay on ECM after incubation with control (VEGF-C “-”) or with VEGF-C [100 ng/ml] (VEGF-C “+”), relative to average tube length of untreated L-LECs transduced with control adenovirus (Slit2N-Adenovirus “-”, VEGF-C “-”). Data represent the mean ± SD of 3 independent experiments (***p < 0.001).

Figure 2

Slit2N attenuates VEGF-C-induced activation of VEGFR-3 in L-LECs. (A) Representative VEGFR-3 IP/Western blot analysis of phosphorylated VEGFR-3 in L-LECs after pretreatment with various concentrations of Slit2N, and incubation with VEGF-C [100 ng/ml]. Total VEGFR-3 used as loading control. (B) Quantitative analysis of VEGFR-3 IP/Western blot analysis of phosphorylated VEGFR-3 in L-LECs after pretreatment with various concentrations of Slit2N, and incubation with VEGF-C. Band intensities from Figure 2A were determined by densitometry. The ratio of p-VEGFR-3/total VEGFR-3 of L-LECs treated with VEGF-C alone (2^nd lanes from the left) was set to “1” and the ratios of all other conditions were calculated vs. this experimental condition. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; **p < 0.01). (C) Representative Western blot analysis of phosphorylated VEGFR-2 in L-LECs after pretreatment with various concentrations of Slit2N, and incubation with VEGF-C [100 ng/ml]. Total VEGFR-2 used as loading control. (D) Quantitative analysis of Western blot analysis of phosphorylated VEGFR-2 in L-LECs after pretreatment with various concentrations of Slit2N, and incubation with VEGF-C. Band intensities from Figure 2C were determined by densitometry. The ratio of p-VEGFR-2/total VEGFR-2 of L-LECs treated with VEGF-C alone (2^nd lanes from the left) was set to “1” and the ratios of all other conditions were calculated vs. this experimental condition. Data represent the mean ± SD of 3 independent experiments.

Figure 3

Effects of Slit2N and VEGF-C on total VEGFR-3 levels and VEGFR-3 surface presentation/internalization in L-LECs. (A) Representative Western blot analysis of surface and total VEGFR-3 in L-LECs after incubation with 10 nM Slit2N for indicated times. GAPDH: loading control. (B) Quantitative analysis of surface VEGFR-3 in L-LECs after incubation with 10 nM Slit2N for indicated times. Band intensities from Figure 3A were determined by densitometry. Ratio of surface VEGFR-3/GAPDH in untreated L-LECs (“0” lanes) was set to “1” and values of other ratios were calculated vs. this control. (C) Quantitative analysis of total VEGFR-3 in L-LECs after incubation with 10 nM Slit2N for indicated times. Band intensities from Figure 3A were determined by densitometry. Ratio of total VEGFR-3/GAPDH in untreated L-LECs (“0” lanes) was set to “1” and values of other ratios were calculated vs. untreated control. (D) Representative Western blot analysis of membrane-bound VEGFR-3 (Surface VEGFR-3) and total VEGFR-3 in L-LECs after incubation with VEGF-C alone [100 ng/ml], and 10 nM Slit2N pretreatment + VEGF-C for indicated times. GAPDH: loading control. (E) Quantitative analysis of surface VEGFR-3 in L-LECs after incubation with VEGF-C alone [100 ng/ml] (Control), and 10 nM Slit2N pretreatment + VEGF-C for indicated times. Band intensities from Figure 3D were determined by densitometry. Ratio of surface VEGFR-3/GAPDH of untreated L-LECs was set to “1” and values of all other ratios were calculated vs. untreated control. (F) Quantitative analysis of total VEGFR-3 in L-LECs after incubation with VEGF-C alone [100 ng/ml] (Control), and 10 nM Slit2N pretreatment + VEGF-C for indicated times. Band intensities from Figure 3D were determined by densitometry. Ratio of total VEGFR-3/GAPDH of untreated L-LECs was set to “1” and values of other ratios were calculated vs. untreated control. Data for (B), (C), (E) and (F) represent the mean ± SD of 3 independent experiments (***p < 0.001).

Figure 4

Slit2N inhibits VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).

Figure 5

Slit2N inhibits VEGF-C-induced activation of VEGFR-3 in 293/VEGFR-3 transfectants and L-LECs in a Robo4-dependent manner. (A) Representative Western blot analysis of Robo1 and Robo4 expression in L-LECs, HMVECs, and 293/VEGFR-3 cells. GAPDH used as loading control. (B) Representative Western blot analysis of Robo4 and Robo1 expression in 293/VEGFR-3 cells, 24 h after transfection with pCMV-RFP (vector) or pCMV-RFP-Robo4 (Robo4). GAPDH used as loading control. (C) Representative VEGFR-3 IP/Western blot analysis of phosphorylated VEGFR-3. 293/VEGFR-3 cells were transfected with pCMV-RFP (vector) or pCMV-RFP-Robo4 (Robo4). After 48 h, cells were incubated with a control (“- -”), 10 nM Slit2N, or VEGF-C [100 ng/ml]; or pretreated with Slit2N, then VEGF-C. Total VEGFR-3 used as loading control. (D) Quantitative analysis by densitometry of Figure 5C. The ratio of p-VEGFR-3/VEGFR-3 in vector-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001). (E) Representative Western blot analysis of Robo4 and Robo1 expression in L-LECs, 24 h after transfection with control siRNAs or Robo4-specific siRNAs. GAPDH used as loading control. (F) Representative VEGFR-3 IP/Western blot analysis of phosphorylated VEGFR-3 in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, after incubation with control, VEGF-C [100 ng/ml]; or pretreatment with 10 nM Slit2N, then VEGF-C. Total VEGFR-3 used as loading control. (G) Quantitative analysis by densitometry of Figure 5F. The ratio of p-VEGFR-3/VEGFR-3 in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (*p < 0.05).

Figure 6

Slit2N inhibition of VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs is Robo4-dependent. (A) PI3K activity by ELISA in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, incubated with control or VEGF-C [100 ng/ml]; or after pretreatment with 10 nM Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). (B) Representative Western blot analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Total Akt used as loading control. (C) Quantitative analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Band intensity of each lane from Figure 6B was determined by densitometry. The ratio of p-Akt/total Akt in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001).

Figure 7

Slit2N inhibits VEGF-C-enhanced growth, migration, and tube formation of L-LECs in a Robo4-dependent manner. (A) Proliferation of L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs as assessed by MTS assay after treatment with control, 10 nM Slit2N, VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (**p < 0.01; NS: not statistically significant). (B) Transwell migration of L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs after treatment with control, 10 nM Slit2N, VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (***p < 0.001; NS: not statistically significant). (C) Relative length of tubes formed by L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs as assessed by in vitro tube formation assay on ECM after treatment with control, 10 nM Slit2N, VEGF-C [100 ng/ml]; or after preincubation with Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). For panels A, B, and C, proliferative index, migration index, and relative tube length, respectively, were set to “1” for control-siRNA-transfected, untreated cells. Data for all other conditions were calculated relative to these controls.