Involvement of lysosomal degradation in VEGF-C-induced down-regulation of VEGFR-3

Abstract

The vascular endothelial growth factor (VEGF)-C-induced down-regulation of VEGF receptor (VEGFR)-3 is important in lymphangiogenesis. Here, we demonstrate that VEGF-C, -D, and -C156S, but not VEGF-A, down-regulate VEGFR-3. VEGF-C stimulates VEGFR-3 tyrosyl phosphorylation and transient phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinases in lymphatic endothelial cells. VEGF-C-induced down-regulation of VEGFR-3 was blocked by a VEGF-C trap, tyrosine kinase inhibitor, and leupeptin, pepstatin, and E64 (LPE), but was unaffected by Notch 1 activator and γ-secretase inhibitors. Our findings indicate that VEGF-C down-regulates VEGFR-3 in lymphatic endothelial cells through VEGFR-3 kinase activation and, in part, via lysosomal degradation.

Keywords: Lysosomal degradation; VEGF receptor 3; VEGF-C.

Figure 1

VEGF-C treatment down-regulates VEGFR-3 protein and mRNA expression in SV-LECs and hLECs. Western blot analysis of VEGFR-3 in: (A) SV-LECs and hLECs left untreated or stimulated with VEGF-C for 24 hours; (B) vECs at the indicated times after VEGF-C (50 ng/mL) stimulation; and (C) serum-starved SV-LECs exposed to increasing concentrations of VEGF-C for 24 hours. (D) Quantitative real-time PCR analysis of VEGFR-2 and -3 as well as GAPDH mRNA levels in SV-LECs stimulated with VEGF-C for 24 hours.

Figure 2

VEGFR-3 protein expression is down-regulated by treatment with VEGF-C and -D, but not VEGF-A, and down-regulation by VEGF-C can be blocked by VEGFR-3-Fc. Western blot analysis of VEGFR-3 in: (A) serum-starved SV-LECs left untreated or stimulated with VEGF-C (C156S, 0–500 ng/mL), VEGF-A (50 ng/mL), or VEGF-D (20–50 ng/mL) for 24 hours; (B) serum-starved SV-LECs untreated or stimulated with VEGF-C for 24 hours with or without the the VEGF-C trap VEGFR-3-Fc (1 μg/mL); and (C) cell lysates and conditioned media collected from SV-LECs stimulated with VEGF-C for 24 hours.

Figure 3

VEGF-C induces tyrosyl phosphorylation of ERK, JNK, and p38 in serum-starved SV-LECs. Western blot analysis of: (A) total (left) and tyrosine phosphorylated (right) VEGFR-3 in SV-LECs left untreated or stimulated with VEGF-C for 10 min; and (B) total (lower rows) and phosphorylated (upper rows) ERK, JNK, and p38 in SV-LECs stimulated with VEGF-C (1 or 50 ng/mL as indicated) for 0, 5, 15, or 30 min.

Figure 4

VEGF-C–induced down-regulation of VEGFR-3 protein expression is not affected by a MMP inhibitor, Notch 1 activator, or γ-secretase inhibitor but is inhibited by AZD1217 and LPE. Western blot analysis of VEGFR-3 in: (A) serum-starved SV-LECs treated with either GM6001 (MMP inhibitor, 100 nM), VEGF-C (50 ng/mL), or both for 24 hours; (B) serum-starved SV-LECs left untreated or stimulated with VEGF-C (50 ng/ml) for 24 hours in the presence or absence of the Notch activator, Jag-1 (1 or 10 μM), or the γ-secretase inhibitor, DAPT (10 μM); (C) SV-LECs left untreated or stimulated with VEGF-C in the presence or absence of the TKI, AZD2171 (10 or 1 nM); and (D) inhibition of VEGF-C–induced VEGFR-3 down-regulation by lysosomal protease inhibitors (LPE) after 6 hours of incubation.