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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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FAK is required for angiogenesis and signaling during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS(A)-FRNK (inactive FAK) or i.v. injected with anti-αvβ3 or -αvβ5 followed by stimulation with either 2 μg/ml 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 20 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-FRNK (inactive FAK) followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. Tissues were then excised and subjected to detergent extraction. Relative Ras activity was determined using a pulldown assay with the Ras-binding domain of c-Raf followed by SDS-PAGE and immunoblotting for Ras as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that c-Raf was immunoprecipitated from the tissue extracts and subjected to an in vitro kinase assay using kinase-dead MEK as a substrate as described in Materials and methods. The above blot was probed with an anti-c-Raf antibody as a loading control. (D) Chick CAMs were treated as above with the exception that total CAM lysates were electrophoresed and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control.
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fig2: FAK is required for angiogenesis and signaling during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS(A)-FRNK (inactive FAK) or i.v. injected with anti-αvβ3 or -αvβ5 followed by stimulation with either 2 μg/ml 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 20 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-FRNK (inactive FAK) followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. Tissues were then excised and subjected to detergent extraction. Relative Ras activity was determined using a pulldown assay with the Ras-binding domain of c-Raf followed by SDS-PAGE and immunoblotting for Ras as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that c-Raf was immunoprecipitated from the tissue extracts and subjected to an in vitro kinase assay using kinase-dead MEK as a substrate as described in Materials and methods. The above blot was probed with an anti-c-Raf antibody as a loading control. (D) Chick CAMs were treated as above with the exception that total CAM lysates were electrophoresed and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control.

Mentions: FAK is stimulated on integrin ligation (Schaller, 2001) and plays a critical role in growth factor signaling (Sieg et al., 2000). To further evaluate the role of integrin signaling in angiogenesis, we asked whether FAK activity was required for angiogenesis and if so, how it might contribute to bFGF- or VEGF-mediated Ras-ERK activation. For this purpose, CAMs stimulated with either bFGF or VEGF were transduced with a retrovirus encoding FAK-related nonkinase (FRNK), an autonomously expressed form of FAK containing the COOH-terminal region of FAK, but lacking its kinase domain. Consistent with analyses using integrin antagonists (Brooks et al., 1994a; Friedlander et al., 1995) and reports examining FAK's role in growth factor–induced ERK activity and migration (Renshaw et al., 1999; Sieg et al., 2000), blockade of FAK activity in these tissues disrupted angiogenesis induced with either growth factor (Fig. 2 A). Although FAK was required for c-Raf and ERK activity in response to either growth factor, only Ras activity downstream of VEGF was FAK dependent (Fig. 2, B–D). These results are consistent with those obtained when anti-αvβ3 and anti-αvβ5 were applied to these tissues. Specifically, VEGF-mediated Ras activity requires FAK and αvβ5, whereas bFGF activation of Ras is both FAK- and αvβ3-independent. However, both growth factors require their respective αv integrin and FAK for activation of c-Raf and ERK leading to 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)

FAK is required for angiogenesis and signaling during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS(A)-FRNK (inactive FAK) or i.v. injected with anti-αvβ3 or -αvβ5 followed by stimulation with either 2 μg/ml 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 20 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-FRNK (inactive FAK) followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. Tissues were then excised and subjected to detergent extraction. Relative Ras activity was determined using a pulldown assay with the Ras-binding domain of c-Raf followed by SDS-PAGE and immunoblotting for Ras as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that c-Raf was immunoprecipitated from the tissue extracts and subjected to an in vitro kinase assay using kinase-dead MEK as a substrate as described in Materials and methods. The above blot was probed with an anti-c-Raf antibody as a loading control. (D) Chick CAMs were treated as above with the exception that total CAM lysates were electrophoresed and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control.
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Related In: Results  -  Collection

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fig2: FAK is required for angiogenesis and signaling during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS(A)-FRNK (inactive FAK) or i.v. injected with anti-αvβ3 or -αvβ5 followed by stimulation with either 2 μg/ml 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 20 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-FRNK (inactive FAK) followed by stimulation with either 2 μg/ml bFGF or VEGF for 20 h. Tissues were then excised and subjected to detergent extraction. Relative Ras activity was determined using a pulldown assay with the Ras-binding domain of c-Raf followed by SDS-PAGE and immunoblotting for Ras as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that c-Raf was immunoprecipitated from the tissue extracts and subjected to an in vitro kinase assay using kinase-dead MEK as a substrate as described in Materials and methods. The above blot was probed with an anti-c-Raf antibody as a loading control. (D) Chick CAMs were treated as above with the exception that total CAM lysates were electrophoresed and probed with antibodies directed against the active phosphorylated form of ERK or an anti-ERK antibody as a loading control.
Mentions: FAK is stimulated on integrin ligation (Schaller, 2001) and plays a critical role in growth factor signaling (Sieg et al., 2000). To further evaluate the role of integrin signaling in angiogenesis, we asked whether FAK activity was required for angiogenesis and if so, how it might contribute to bFGF- or VEGF-mediated Ras-ERK activation. For this purpose, CAMs stimulated with either bFGF or VEGF were transduced with a retrovirus encoding FAK-related nonkinase (FRNK), an autonomously expressed form of FAK containing the COOH-terminal region of FAK, but lacking its kinase domain. Consistent with analyses using integrin antagonists (Brooks et al., 1994a; Friedlander et al., 1995) and reports examining FAK's role in growth factor–induced ERK activity and migration (Renshaw et al., 1999; Sieg et al., 2000), blockade of FAK activity in these tissues disrupted angiogenesis induced with either growth factor (Fig. 2 A). Although FAK was required for c-Raf and ERK activity in response to either growth factor, only Ras activity downstream of VEGF was FAK dependent (Fig. 2, B–D). These results are consistent with those obtained when anti-αvβ3 and anti-αvβ5 were applied to these tissues. Specifically, VEGF-mediated Ras activity requires FAK and αvβ5, whereas bFGF activation of Ras is both FAK- and αvβ3-independent. However, both growth factors require their respective αv integrin and FAK for activation of c-Raf and ERK leading to 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