The docking protein FRS2α is a critical regulator of VEGF receptors signaling

Abstract

Vascular endothelial growth factors (VEGFs) signal via their cognate receptor tyrosine kinases designated VEGFR1-3. We report that the docking protein fibroblast growth factor receptor substrate 2 (FRS2α) plays a critical role in cell signaling via these receptors. In vitro FRS2α regulates VEGF-A and VEGF-C-dependent activation of extracellular signal-regulated receptor kinase signaling and blood and lymphatic endothelial cells migration and proliferation. In vivo endothelial-specific deletion of FRS2α results in the profound impairment of postnatal vascular development and adult angiogenesis, lymphangiogenesis, and arteriogenesis. We conclude that FRS2α is a previously unidentified component of VEGF receptors signaling.

Keywords: FGF receptor; MAP kinase; phosphorylation; receptor kinase inhibition; signal transduction.

Fig. 1.

FRS2α knockdown in HUVEC inhibits VEGF-A₁₆₅–dependent signaling. (A) Control and FRS2α knockdown HUVEC were serum starved overnight and treated with VEGF-A₁₆₅ (50 ng/mL) as indicated. Cell lysates were immunoprecipitated (IP) with an anti-VEGFR2 antibody and immunoblotted with anti–p-Tyrosine antibody. The same blot was stripped and blotted with anti-VEGFR2. Input lysates were blotted with anti-VEGFR2 and anti-FRS2α antibodies. (B) Control and FRS2α knockdown HUVEC were serum starved overnight and treated with VEGF-A₁₆₅ (50 ng/mL) as indicated. Cell lysates were blotted with p-VEGFR2, VEGFR2, p-ERK, ERK, and FRS2α antibodies. (C) Control and FRS2α-6F (Flag) overexpressed HUVEC were serum starved overnight and treated with VEGF-A₁₆₅ (50 ng/mL). Cell lysates were blotted with p-VEGFR2, VEGFR2, p-ERK, ERK, and Flag (FRS2α) antibodies. (D) Control and FRS2α knockdown HUVEC were serum starved overnight. Cell proliferation (Left) and cell migration (Right) in response to VEGF-A₁₆₅ (50 ng/mL) profiles are shown as detected by xCELLigence. Approximately 1000 cells (for cell proliferation) or 25,000 cells (for cell migration) were loaded per well in duplicate (***P < 0.01 compared with control). (E) In vitro Matrigel: The extent of cords branching was assessed in control and FRS2α knockdown HUVEC placed on growth factor-depleted Matrigel and exposed to VEGF-A₁₆₅ (50 ng/mL). Data in A–C and E are based three independent experiments; data in D are based on two independent experiments.

Fig. 2.

FRS2α knockdown in HDLEC inhibits VEGF-C–dependent signaling. (A) Control and FRS2α knockdown HDLEC were serum starved overnight and treated with VEGF-C (50 ng/mL) for the indicated times. Cell lysates were immunoprecipitated (IP) with an anti-VEGFR3 antibody and immunoblotted with anti–p-Tyrosine antibody. The same blot was stripped and blotted with anti-VEGFR3. Input lysates were blotted with anti-VEGFR3 and anti-FRS2α antibodies. (B Upper) Control and FRS2α knockdown HDLEC were serum starved overnight and treated with VEGF-C (50 ng/mL). Cell lysates were blotted with p-ERK, ERK, and FRS2α antibodies. (B Lower) Control and FRS2α6F (Flag) overexpressed HDLEC were serum starved overnight and treated with VEGF-C (50 ng/mL). Cell lysates were blotted with p-ERK, ERK, and FRS2α antibodies. (C) Control and FRS2α knockdown HDLEC were serum starved overnight. Cell proliferation (Upper) and cell migration (Lower) in response to VEGF-C (50 ng/mL) profiles, detected by xCELLigence. Approximately 1,000 cells (for cell proliferation) or 25,000 cells (for cell migration) were loaded per well in duplicate (***P < 0.01 compared with control). (D) Control and FRS2α knockdown HUVEC were serum starved overnight and treated with PlGF-1 (50 ng/mL). Cell lysates were immunoprecipitated (IP) with an anti-VEGFR1 antibody and immunoblotted with anti–p-Tyrosine antibody. The same blot was stripped and blotted with anti-VEGFR1. Input lysates were blotted with anti-VEGFR1 and anti-FRS2α antibodies. Data shown in A, B, and D are based on three independent experiments; data in C are based on two independent experiments.

Fig. 3.

Impaired angiogenesis in Frs2α^−/− mice. (A) Wild-type and Frs2α^−/− mice were treated with 1 × 10⁹ pfu of Ad-LacZ or Ad-VEGF-A₁₆₄ virus. VEGF-A–induced angiogenesis was recorded at day 7 by using a stereomicroscope and fluorescent scope. (B) Mouse ears were sectioned, and the number of vessels was counted (**P < 0.01 compared with control) (n = 4 mice per group). (C and D) Matrigel mixed with either PBS or VEGF-A₁₆₅ (50 ng/mL) were placed s.c. in wild-type or Frs2α^−/− mice. On day 7, matrigel plugs were sectioned, and the number of vessels was counted (*P < 0.05 compared with control) (n = 6 mice per group). (E–H) Hydron pellet containing VEGF-A₁₆₄ or VEGF-C was implanted into the cornea of wild-type and Frs2α^−/− mice. Angiogenesis was assessed by stereo microscopy at day 7 following implantation (E and G) (asterisk marks the position of the implanted pellet). Vascular density was quantified in F and H (*P < 0.05; **P < 0.01 compared with control) (n = 6 mice per group).

Fig. 4.

Impaired arteriogenesis in Frs2α^−/− mice. (A) Laser Doppler images showing blood flow before and after the induction of ischemia to the left hindlimb in wild-type and Frs2α^−/− mice. (B) Laser-Doppler analysis of blood flow recovery in the left foot, expressed as a ratio of blood flow in left to right foot (L/R). *P < 0.05, wild-type vs. Frs2α^−/− (n = 8 mice per group). (C) In Frs2α^−/− mice, clinical score indicated a severe phenotype, leading to necrosis of limb. (D) Representative sections from nonischemic and ischemic groups of wild-type and Frs2α^−/− on day 14 after ischemia. Quantification of capillary density (number/mm² muscle area) and ratio of CD31/myocyte are shown in E. Data are mean ± SD from 10 fields per section (3 sections per mouse; n = 4 for each strain). *P < 0.05, wild-type vs. Frs2α^−/−.

Fig. 5.

Postnatal angiogenesis and lymphangiogenesis defects in Frs2α^−/− mice. (A–D) P6 retinas from wild-type and Frs2α^−/− littermate mice. (E and F) P6 diaphragms from wild-type and Frs2α^−/− littermate mice. a’ and b’ panels show higher magnification of the angiogenic front (A–D) and diaphragm lymphatic vessels (E and F).