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Differential alphav integrin-mediated Ras-ERK signaling during two pathways of angiogenesis.

Hood JD, Frausto R, Kiosses WB, Schwartz MA, Cheresh DA - J. Cell Biol. (2003)

Bottom Line: Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation.The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak.Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Antagonists of alphavbeta3 and alphavbeta5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these alphav integrins differentially contribute to sustained Ras-extracellular signal-related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-alphavbeta5 blocked upstream of Ras, whereas anti-alphavbeta3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.

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PAK activity is required for bFGF-mediated ERK activation and angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. CAM tissue was excised, subjected to detergent extraction, electrophoresed, and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control as described in Materials and methods. (B) Chick CAMs were treated as above with the exception that after 20 h, the angiogenic tissue was resected and snap frozen. Tissue sections were probed with an antibody directed against the active phosphorylated form of ERK. (C) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either bFGF or VEGF for 72 h. Blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 36 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment.
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fig6: PAK activity is required for bFGF-mediated ERK activation and angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. CAM tissue was excised, subjected to detergent extraction, electrophoresed, and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control as described in Materials and methods. (B) Chick CAMs were treated as above with the exception that after 20 h, the angiogenic tissue was resected and snap frozen. Tissue sections were probed with an antibody directed against the active phosphorylated form of ERK. (C) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either bFGF or VEGF for 72 h. Blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 36 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment.

Mentions: Consistent with these results, transduction of CAMs with an RCAS retrovirus encoding the PAK-1 auto-inhibitory domain (PAK83–149; Zhao et al., 1998) selectively disrupted bFGF-induced ERK activity (Fig. 6 A) in these tissues. This finding was confirmed in situ, using an antibody specific for phosphorylated (activated) ERK, which revealed that the suppression of PAK-1 selectively blocked bFGF-mediated ERK activation within angiogenic blood vessels (Fig. 6 B). These findings were extended by evaluating angiogenesis on CAMs stimulated with VEGF- and bFGF-mediated or bFGF and transduced with RCAS-PAK83–149. Although both growth factors stimulated angiogenesis, inhibition of PAK-1 selectively suppressed bFGF-induced angiogenesis (Fig. 6 C). These findings reveal that integrin αvβ3 regulates PAK-1 activity during bFGF-induced angiogenesis in vivo, and that PAK-1 selectively mediates bFGF-induced ERK activation and angiogenesis.


Differential alphav integrin-mediated Ras-ERK signaling during two pathways of angiogenesis.

Hood JD, Frausto R, Kiosses WB, Schwartz MA, Cheresh DA - J. Cell Biol. (2003)

PAK activity is required for bFGF-mediated ERK activation and angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. CAM tissue was excised, subjected to detergent extraction, electrophoresed, and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control as described in Materials and methods. (B) Chick CAMs were treated as above with the exception that after 20 h, the angiogenic tissue was resected and snap frozen. Tissue sections were probed with an antibody directed against the active phosphorylated form of ERK. (C) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either bFGF or VEGF for 72 h. Blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 36 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment.
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Related In: Results  -  Collection

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fig6: PAK activity is required for bFGF-mediated ERK activation and angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. CAM tissue was excised, subjected to detergent extraction, electrophoresed, and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control as described in Materials and methods. (B) Chick CAMs were treated as above with the exception that after 20 h, the angiogenic tissue was resected and snap frozen. Tissue sections were probed with an antibody directed against the active phosphorylated form of ERK. (C) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-PAK83–149 (inactive PAK), followed by stimulation with either bFGF or VEGF for 72 h. Blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 36 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment.
Mentions: Consistent with these results, transduction of CAMs with an RCAS retrovirus encoding the PAK-1 auto-inhibitory domain (PAK83–149; Zhao et al., 1998) selectively disrupted bFGF-induced ERK activity (Fig. 6 A) in these tissues. This finding was confirmed in situ, using an antibody specific for phosphorylated (activated) ERK, which revealed that the suppression of PAK-1 selectively blocked bFGF-mediated ERK activation within angiogenic blood vessels (Fig. 6 B). These findings were extended by evaluating angiogenesis on CAMs stimulated with VEGF- and bFGF-mediated or bFGF and transduced with RCAS-PAK83–149. Although both growth factors stimulated angiogenesis, inhibition of PAK-1 selectively suppressed bFGF-induced angiogenesis (Fig. 6 C). These findings reveal that integrin αvβ3 regulates PAK-1 activity during bFGF-induced angiogenesis in vivo, and that PAK-1 selectively mediates bFGF-induced ERK activation and angiogenesis.

Bottom Line: Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation.The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak.Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Antagonists of alphavbeta3 and alphavbeta5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these alphav integrins differentially contribute to sustained Ras-extracellular signal-related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-alphavbeta5 blocked upstream of Ras, whereas anti-alphavbeta3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.

Show MeSH
Related in: MedlinePlus