Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase - PubMed (original) (raw)

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase

Shideh Kazerounian et al. Blood. 2011.

Abstract

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

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Figures

Figure 1

Association of CD36 with integrins and tetraspanins. HDMECs were lysed in 1% Brij 99 (supplemental Figures 1-2). One milligram of protein was immunoprecipitated with a combination of 3 μg of FA6-152 and 2 μg of CRF-2712 anti-CD36 Abs (lane 2) or 5 μg of nonimmune mouse Ab (lane 1). The immunoprecipitants were Western blotted with Abs against (A) identified integrin subunits in Ab-array assays in supplemental Figure 1B or in platelets and (B) tetraspanin family members. (C) To address the role of CD9 and CD151 as linkers for the association of integrin β1 with CD36, lungs from CD151- and CD9-null mice were isolated, and proteins were extracted, as described in the immunoprecipitation section in “Methods.” Anti-CD36 Abs (lane 2) or nonimmune IgG (lane 1) immunoprecipitants were probed for anti-integrin β1 Abs. The lack of CD9 or CD151 did not abolish the CD36-β1 interaction suggesting that either these 2 tetraspanins can compensate for each other, or there is a direct interaction between CD36 and β1, as proposed by others.,

Figure 2

Interaction of CD36 with Syk, VEGFR2, and their downstream signaling proteins. HDMECs were lysed either in 1% Brij 99 or 1% Triton X-100, and an equal amount of protein was used for immunoprecipitation. (A) Anti-CD36 (lane 2) and nonimmune IgG (lane 1) immunoprecipitants were Western blotted with Abs to Syk, Vav, and p85 PI3K. The p85 subunit of PI3K was detected in 1% Brij 99, while Syk and Vav were present in 1% Triton X-100 immunoprecipitants (supplemental Figure 1B). (B) Triton X-100–solubilized lysates were immunoprecipitated with anti-Syk (lane 2) and nonimmune IgG (lane 1) Abs (supplemental Figure 1B). The nitrocellulose membrane was probed with an anti-CD36 Ab. (C) In reciprocal immunoprecipitations, HDMEC cells were lysed in 1% Brij 99 and immunoprecipitated with anti-VEGFR-2 (lane 2) and nonimmune IgG (lane 1) Abs, and immunoprecipitants were probed with either anti-Syk (left) or anti-CD36 (right) Abs (supplemental Figure 3). (D-E) To determine whether Triton X-100 can disrupt the association of Syk and CD36 with VEGFR-2, HDMECs were lysed in either 1% Brij 99 or 1% Triton X-100 and immunoprecipitated with anti-VEGFR-2 (lane 2) and nonimmune IgG (lane 1) Abs. Immunoprecipitants were probed with anti-Syk, anti-CD36, and anti-VEGFR-2 Abs, as indicated.

Figure 3

Association of CD36 with VEGFR-2, Syk, and integrin β1 subunit in the lungs of wild-type and TSP-1--null mice. (A) Brij 99 lysates from TSP-1–null and wild-type lung tissues were immunoprecipitated with nonimmune IgG (lane 1) or anti-CD36 Ab (lane 2) and Western blotted with anti-β1 integrin Ab. (B) Top: Triton X-100 lysates from TSP-1–null and wild-type lung tissues were immunoprecipitated with nonimmune IgG (lane 1) or an anti-Syk Ab (lane 2) and Western blotted with an anti-CD36 Ab. Middle: In parallel experiments, tissue extracts were immunoprecipitated with anti-CD36 Ab (lane 2) and nonimmune IgG (lane 1) and Western blotted with anti-Syk and anti-CD36 Abs, as indicated. Bottom: The high-density fractions of sucrose gradients prepared with Triton X-100 extracts of TSP-1-null and wild-type lung tissues (supplemental Figure 6B) were immunoprecipitated with nonimmune IgG (lane 1) or anti-CD36 Ab (lane 2) and Western blotted with an anti-Syk Ab. (C) Top: Brij 99 lysates from TSP-1–null and wild-type lung tissues were immunoprecipitated with nonimmune IgG (lane 1) or anti-CD36 Ab (lane 2) and Western blotted with anti-VEGFR2 Ab. In this experiment, the total lysates were used as loading control (middle panel). Bottom: Brij 99 lysates from TSP-1–null and wild-type lung tissues were immunoprecipitated with nonimmune IgG (lane 1) or anti-VEGFR-2 Ab (lane 2) and Western blotted with anti-CD36 Ab. The immunoprecipitates were also Western blotted with an anti-VEGFR-2 Ab to establish that comparable amounts of VEGFR-2 were immunoprecipitated from the TSP-1–null and wild-type tissue. (D) Top: Brij 99 lysates from TSP-1–null and wild-type lung tissue were immunoprecipitated with nonimmune IgG (lane 1) or anti-Syk Ab (lane 2) and Western blotted with anti-VEGFR-2 or anti-Syk Abs, as indicated. Bottom: Brij 99 lysates from TSP-1–null and wild-type lung tissue were immunoprecipitated with nonimmune IgG (lane 1) or anti-VEGFR-2 Ab (lane 2) and Western blotted with anti-Syk and anti-VEGFR-2 Abs. In these experiments, either total lysates or immunoprecipitants were also Western blotted to establish that comparable amount of proteins were detected or immunoprecipitated from TSP-1–null and wild-type tissues.

Figure 4

VEGF-A–induced phosphorylation of Syk (Y323). (A) HDMECs were serum-starved overnight in media containing 2% FBS. Cells were washed and incubated with either 0.5% FBS in PBS or treated with VEGF-A (50 ng/mL) in 0.5% FBS in PBS for 10 minutes at 37°C. Cells were lysed in 1% Triton X-100 lysis buffer, and equal amounts of protein were Western blotted with anti-p-Y323 Syk, total Syk (supplemental Figure 5A), anti-p-Y1175 VEGFR-2, and total VEGFR-2 Abs, as indicated. The results showed an up-regulation of p-Y323 Syk in response to VEGF-A treatment. (B) Parallel studies were performed using HUVECs that were infected with full-length CD36 or empty (PINCO) vector (supplemental Figure 4B). After treatment of cells with VEGF-A, cells were lysed in 1% Triton X-100, and immunoblotting was performed as indicated. A significant increase in the level of p-Y1175 VEGFR-2 and p-Y323 Syk was only detected in HUVECs with overexpression of CD36. Note that Syk expression is significantly higher in the HUVECs that were engineered to express CD36 (bottom panel, total Syk, and supplemental Figure 4B). These experiments were repeated 3 times independently. (C) Wild-type and TSP-1–null mice were injected with 2 μg of VEGF-A in 100 μL of sterile PBS via the tail vein. After 5 minutes, mice were euthanized and lungs were harvested as described in the immunoblotting section in “Methods.” An equal amount of protein was Western blotted with anti-p-Y323 and anti-total Syk. VEGF-A treatment up-regulated the level of Syk phosphorylation as was seen in panel A, but the response was higher in wild-type mice compared with TSP-1–null, even though the wild-type mice express lower levels of total Syk (bottom panel and supplemental Figure 4A). The slight difference in Syk expression (Figure 4A-B) is possibly because of difference in loading, as it is also detected in actin and VEGFR-2 lanes. Because cells were treated only for 10 minutes, it is not sufficient time for VEGF-A to up-regulate Syk expression. In these experiments, we resuspended cells in PBS with BSA to avoid any interference from the media or serum.

Figure 5

Reduction of VEGF-A–induced p-Y1175 VEGFR-2 by a Syk inhibitor. (A) HDMECs were serum-starved overnight and washed with PBS (supplemental Figure 5A). Cells were incubated with 0.5% FBS in PBS with or without 4.7μM BAY 61-3606 (Syk inhibitor), as described in the endothelial kinase assay section in “Methods.” Cells were lysed in 1% Triton X-100, and an equal amount of protein was used for immunoblotting using Abs against p-Y323 Syk, or p-1054 VEGFR2 or p-Y1175 VEGFR-2. Inhibition of Syk did not affect the autophosphorylation of VEGFR-2 at Y1054, but it decreased phosphorylation of Y1175 in response to VEGF-A. (B) The experiment above was repeated using HUVECs that were infected with either control vector or full-length CD36 vector (supplemental Figure 4B). Cells were treated as described in panel A and phosphorylation of Syk and VEGFR-2 was detected. The results showed that Syk inhibitor reduced phosphorylation of VEGFR-2 at Y1175, but did not affect the level of p-Y1054 in cells with overexpression of CD36. The level of both p-Y1175 and p-Y1054 remained unchanged in control cells. These experiments were done independently 3 times.

Figure 6

Participation of Syk in the VEGFR-2 signaling pathway. (A-B) Syk-dependent migration of HUVECs and HDMECs in response to VEGF-A. HUVECs were infected with either control (PINCO) or full-length CD36 vector (supplemental Figure 4B-C). Cells were serum-starved, and some were treated with 4.7μM Syk inhibitor (BAY 61-3606) while others were left untreated, as described in the endothelial kinase assay section in “Methods.” Results are expressed as a percentage of migrated cells (mean ± SE, n = 8) in 2 independent experiments. P values were calculated with an unpaired Student t test against HUVECs or HDMECs with Syk inhibitor or without inhibitor and VEGF-A. In these experiments, we resuspended cells in PBS with BSA to avoid any interference from the media or serum. (C) A proposed model for the TSP-1 regulation of the VEGF-A–induced VEGFR-2 signaling pathway. (a) Binding of TSP-1 to CD36 and/or VEGFR-2, and their associated integrins and tetraspanins (not shown) brings the 2 receptors in close proximity. (b) VEGF-A initiates angiogenesis by binding to VEGFR-2 and inducing its autophosphorylation, which, ultimately promotes Syk phosphorylation directly or through other adaptor proteins. (c) Syk activation further phosphorylates VEGFR-2 at Y1175 and mediates endothelial cell migration. (d) In the absence of VEGF-A, the TSP-1-CD36 complex only promotes Fyn activation, which inhibits endothelial cell migration and returns the angiogenic switch to the “off” position. The dashed lines suggest the possible involvement of other signaling and adaptor proteins.

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Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase - PubMed (original) (raw)