 |
8, 12, 1155–1161 (2007)
doi:10.1038/sj.embor.7401103
Figures and Tables
Ligand-independent activation of vascular endothelial growth factor receptor 1 by low-density lipoprotein
Ryosuke Usui, Masabumi Shibuya, Shun Ishibashi & Yoshiro Maru
| |
| | |
| |
|
| | |
| | Figures | |
| |
|
|
Figure 1
Native low-density lipoprotein induces endocytosis of VEGFR1. (A) Immunoblotting (IB) of whole-cell lysates (WCLs; 30  g each) from human 293T cells (left) with human LDLR antibody and those from rodent cells with mouse LDLR antibody, including the liver of LDLR-
/-
and WT mice, RAW, CHO9, NIH3T3 cells overexpressing VEGFR1 (3T3/VEGFR1) and peritoneal macrophages (middle). Cell lines were cultured in 1%
FCS for 24 h before protein extraction. WCLs from 3T3/VEGFR1 cultured in 10%
and 1%
serum, or LPDS were also subjected to anti-LDLR and anti-actin immunoblotting (right). (B) WCLs of 293T cells transfected with the VEGFR1-GFP expression vector or mock (-
) were immunoblotted with GFP antibody (left). Membrane localization of VEGFR1-GFP transfected into 293T or RAW cells before nLDL stimulation (right). Scale bars, 10 m. (C,D) 293T or RAW cells transfected with VEGFR1-GFP (green) were incubated with 10 g/ml DiI-nLDL (red) (C) or 10 g/ml DiI-acLDL (red) (D) for 30 min. Note the merged images with nLDL but not acLDL. Scale bars, 10 m. (E) Peritoneal macrophages (M ) derived from WT, tk-
/-
, LDLR-
/-
and RAW cells were immunostained with VEGFR1 and LDLR antibodies before (-
) and after (+) stimulation by nLDL at 100 g/ml. acLDL, acetylated LDL; GFP, green fluorescent protein; LDL, low-density lipoprotein; LDLR, LDL receptor; LPDS, lipoprotein-deficient serum; nLDL, native LDL; VEGFR, vascular endothelial growth factor receptor; WT, wild type.
|
|
Figure 2
Enhanced recruitment of the low-density lipoprotein receptor with VEGFR1 by native LDL stimulation. (A) Whole-cell lysates (WCLs) and anti-VEGFR1 immunoprecipitates (IP) from RAW cells, with (+nLDL) or without (-
nLDL) stimulation by nLDL, were immunoblotted (IB) with LDLR, VEGFR1 and actin antibodies. (B) Anti-LDLR IP and anti-VEGFR1 IP from HUVEC, with (+nLDL) or without (-
nLDL) stimulation by nLDL, were immunoblotted with LDLR or VEGFR1 antibody. (C,D) WCLs (60 g each) and IPs by VEGFR (C) carboxy-terminal antibody (VEGFR1-C) or (D) amino-terminal antibody (VEGFR1-N) from lung lysates of WT or tk-
/-
mice were immunoblotted with LDLR, EGFR, PDGFR- and the corresponding VEGFR1 antibody. EGFR, epidermal growth factor receptor; HUVEC, human umbilical vein endothelial cells; LDL, low-density lipoprotein; LDLR, LDL receptor; nLDL, native LDL; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor; WCL, whole-cell lysate; WT, wild type.
|
|
|
Figure 3
Native low-density lipoprotein induces autophosphorylation of VEGFR1. (A) 3T3/VEGFR1 cells untreated (-
) or pretreated with a VEGFR tyrosine kinase inhibitor SU5416 at 1 M or Src inhibitor PP2 at 2 M were stimulated by VEGF, GA or nLDL for 10 min. WCLs were immunoblotted (IB) with phosphotyrosine (PY), VEGFR1 and actin antibody. (B) WCLs or anti-VEGFR2 immunoprecipitates (IP) from 3T3/VEGFR2 cells stimulated with VEGF or nLDL were immunoblotted with PY, VEGFR2 and actin antibody. (C) WCLs from 3T3/VEGFR1 cells stimulated by the indicated concentration of nLDL after culture in 1%
FCS or LPDS for 24 h were subjected to anti-PY, anti-VEGFR1 and anti-actin immunoblotting. (D) 3T3/VEGFR1 cells pretreated with mock (-
), anti-LDLR siRNAs (antisense/sense: AS) or control siRNAs (sense/sense: SS or irrelevant siRNA against Nox1: AS*) at 40 nM were stimulated by nLDL for 10 min and then subjected to anti-PY, anti-VEGFR1, anti-LDLR and anti-actin immunoblotting. Relative average values of the band intensities from three independent experiments are shown below the western blot panels. (E,F) WCLs and (E) anti-PLC or (F) anti-c-Cbl IP from 3T3/VEGFR1 cells stimulated with VEGF or nLDL were immunoblotted with VEGFR1, PY, PLC (E), c-Cbl (F) and actin antibody. 3T3/VEGFR1, NIH3T3 cells that overexpress VEGFR1; GA, geldanamycin; LPDS, lipoprotein-deficient serum; nLDL, native LDL; PLC, phospholipase C; siRNA, short interfering RNA; VEGFR, vascular endothelial growth factor receptor; WCL, whole-cell lysate.
|
|
Figure 4
Native low-density lipoprotein-induced macrophage migration is VEGFR1-dependent. VEGF or nLDL at the indicated concentrations was added to the lower chambers. Data were corrected for background intensities with no stimulation in WT mice and expressed as means s.d. *P<0.001. Assays were carried out in triplicate. hpf, high-power field; nLDL, native LDL; LDLR, LDL receptor; VEGFR, vascular endothelial growth factor receptor; WT, wild type.
|
|
|
| | |
|
