Anastellin, the angiostatic fibronectin peptide, is a selective inhibitor of lysophospholipid signaling

Abstract

Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein-coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)-dependent G(1)-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G(1)-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a G(i)-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through G(i), anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of G(i) and unaffected by anastellin. These results suggest that lysophospholipid mediators of G(i) activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of G(i) and that anastellin selectively inhibits the Ras/ERK arm of the pathway.

FIGURE 1. LPA and S1P stimulate ERK-dependent proliferation of microvessel endothelial cells

Serum starved human dermal microvessel endothelial cells were treated with LPA (A) or S1P (B) as indicated for 16 h and assayed for incorporation of [³H]-thymidine as described under Materials and Methods. In panel C, serum starved MVECs were treated with the MEK inhibitor PD98059 as indicated for 30 min prior to the addition of either 20 µM LPA or 1 µM S1P and assayed for [³H]-Thymidine incorporation. Alternatively, serum starved MVECs treated with PD98059 as indicted were stimulated with either 20 µM LPA or 1 µM S1P for 6 min (D). Cell lysates were subjected to SDS-PAGE and immunoblotted for changes in phospho-ERK. Stripped membranes were reprobed with an anti-ERK2 antibody for loading control. Statistical differences were measured by comparison with unstimulated cells. *p<0.05; **p< 0.01; ***p <0.001.

FIGURE 2. Lysophospholipid-stimulated ERK activation is independent of Rho signaling

Extracts of MVECs treated with various concentrations of LPA and S1P as indicated for 6 min were subjected to SDS-PAGE followed by immunodetection with antibodies directed against phosphorylated Thr18/Ser19 of myosin light chain 2 (pMLC2), pS473Akt and pERK (A). Alternatively, cells were treated with 1 ng/ml PTx for 20 h (B), 2 µM Y27632, a specific inhibitor of Rho-associated kinase (ROCK) or 5 µM U0126 for 30 min (C) prior to stimulation with 40 µM LPA or 4 µM S1P for 6 min. Cell extracts were subjected to SDS-PAGE and immunoblots probed as described above. Membranes were stripped and reprobed with an anti-ERK2 antibody.

FIGURE 3. Anastellin inhibits lysophospholipid-mediated ERK activation and proliferation in MVECs

Serum starved cells were treated with increasing concentrations of anastellin for 60 min prior to stimulation with 20 µM LPA or 1 µM S1P for 6 min (A). In panel B, cells were treated with 20 µM anastellin for up to 60 min prior to the addition of 20 µM LPA or 1 µM S1P, lysed, and proteins immunoblotted for changes in phospho-ERK. Membranes were stripped and reprobed with an anti-ERK2 antibody. An unrelated type III module of FN (III₁₃) was used as negative control (A). To evaluate the effect of anastellin on lysophospholipid-mediated endothelial cell proliferation, serum starved MVECs were treated with anastellin for 60 min prior to 20 µM LPA and 1 µM S1P stimulation and assessed for [³H]-thymidine incorporation (C) as described in Materials and Methods. Statistical differences were measured by comparison with cells in the absence of III_1C. *p<0.05; **p<0.01; ***p<0.001.

FIGURE 4. Anastellin inhibits lysophospholipid signaling to ERK upstream of Ras

Adherent microvessel endothelial cells were infected with adenoviral constructs of GFP or dominant negative Ras (RasN17) under serum-free conditions for 24 h. Infected cells were then treated with 20 µM LPA or 1 µM S1P for 6 min. Cell lysates were prepared and immunoblotted with phospho-specific antibodies to Akt and ERK. Antibodies directing against the hemaglutinin epitope (HA) were used to detect adenoviral expression of HA-tagged RasN17. Membranes were stripped and reprobed with an anti-ERK2 antibody (A). In panel B, the effect of anastellin on Ras activation by LPA, S1P and EGF was examined by active Ras ELISA as described under Materials and Methods. Statistical differences were measured by comparison with the untreated control cells (a) or with stimulated cells in the absence of III_1C. *p<0.05; **p<0.01; ***p<0.001.

FIGURE 5. Effect of anastellin on lysophospholipid-mediated Rho activation

Serum starved MVECs were treated with 20 µM anastellin or 20 µM III₁₃ as control for 60 min prior to stimulation with either 40 µM LPA or 1 µM S1P for 6 min. Cells were lysed and extracts subjected to SDS-PAGE followed by immunodetection with antibodies directed against pMLC2, pS473Akt, and pERK (A). Membranes were stripped and reprobed with an anti-ERK2 antibody. In panel B, serum starved endothelial cells were treated with 2 µM Y27632 (30 min) or 20 µM anastellin (60 min) prior to 1 h stimulation with 20 µM LPA. Cells were fixed and immuno-stained with phospho-specific antibodies to MLC2 (green). F-actin was visualized with Alexa Fluor 594-conjugated phalloidin (red). LPA-induced formation of cortical stress fibers (arrows) and regions of pMLC2 colocalization (arrow heads) are indicated.

FIGURE 6. Lysophospholipid-mediated endothelial cell migration is PI3-kinase-dependent

Microvessel endothelial cells were seeded onto collagen-coated tissue culture inserts and stimulated with LPA or S1P as indicated (A). Alternatively, cells were treated with either 25 ng/ml PTx (B), 2 µM Y27632 (C), 5 µM U0126 (D), 10 μM LY294002 or 10 µM AktI-1/2 (E), or 20 µM anastellin (F) for 30–60 min in suspension prior to seeding and stimulation with 20 µM LPA or 1 µM S1P. After 4 h, migratory cells were fixed and stained with Hoechst dye, visualized with a digital fluorescent microscope, and counted. The number of migratory cells/10x field obtained from 2–5 independent experiments carried out in triplicate were normalized to untreated control cells. Statistical differences were measured by comparison with cells in the absence of inhibitor. *p<0.05; **p<0.01; ***p<0.001.

FIGURE 7. Lysophospholipid-mediated signaling pathways in MVECs

LPA and S1P activate specific G-protein-coupled receptors (LPA_1–4 and S1P_1–5, respectively) and initiate Ras/ERK, PI3K/Akt, and Rho/ROCK signaling pathways in microvessel endothelial cells. Results presented here have demonstrated that both Ras/ERK and PI3K/Akt signaling is mediated by a G_i-dependent pathway while Rho signaling occurs through an alternate G-protein, possibly G_12/13 or G_q as has been shown in previous studies (–9). In addition, G_i-dependent activation of Ras/ERK and PI3K/Akt occur through independent pathways and are unaffected by inhibition of Rho signaling. These results are further supported by the finding that anastellin suppresses Ras activation by LPA and prevents ERK activation without affecting PI3K/Akt or Rho/ROCK signaling. The dashed line linking S1P receptors to G_12/13 indicates that a weak induction by S1P was observed in MVECs.