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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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Integrins αvβ3 and αvβ5 differentially influence PAK activity during angiogenesis. (A) 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. 1 h before tissue excision, function-blocking antibodies directed against integrin αvβ3 or αvβ5 were i.v. injected. Endogenous PAK was immunoprecipitated from equivalent amounts of total protein and subjected to a kinase assay using myelin basic protein as a substrate, electrophoresed, and transferred to nitrocellulose as described in Materials and methods. The above blot was probed with an anti-PAK antibody as a loading control. (B) Chick CAMs were treated as above with the exception that total lysates were probed with an antibody directed specific to c-Raf phosphorylated at serine 338. The above blot was probed with an anti-c-Raf antibody as a loading control. (C) 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 c-Raf phosphorylated at serine 338. Bar, 50 μm.
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fig5: Integrins αvβ3 and αvβ5 differentially influence PAK activity during angiogenesis. (A) 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. 1 h before tissue excision, function-blocking antibodies directed against integrin αvβ3 or αvβ5 were i.v. injected. Endogenous PAK was immunoprecipitated from equivalent amounts of total protein and subjected to a kinase assay using myelin basic protein as a substrate, electrophoresed, and transferred to nitrocellulose as described in Materials and methods. The above blot was probed with an anti-PAK antibody as a loading control. (B) Chick CAMs were treated as above with the exception that total lysates were probed with an antibody directed specific to c-Raf phosphorylated at serine 338. The above blot was probed with an anti-c-Raf antibody as a loading control. (C) 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 c-Raf phosphorylated at serine 338. Bar, 50 μm.

Mentions: The finding that αvβ3 appears to regulate c-Raf, but not Ras activity in response to bFGF prompted us to evaluate how αvβ3 contributes to c-Raf activation. Recently, we found that bFGF-mediated angiogenesis is dependent on PAK-1 signaling (Kiosses et al., 2002). Importantly, PAK-1 phosphorylates c-Raf on serine 338, leading to its activation (Zang et al., 2002). To evaluate whether αvβ3 signaling is linked to PAK-1's role in angiogenesis, CAMs stimulated with bFGF or VEGF and treated with anti-αvβ3 or -αvβ5 were lysed and analyzed for PAK-1 activity. Although both growth factors stimulated PAK activity, only anti-αvβ3 was able to suppress this response (Fig. 5 A). Accordingly, direct evaluation of the tissue lysates revealed that bFGF (but not VEGF) induced an αvβ3-dependent phosphorylation of c-Raf at its PAK phosphorylation site, S338 (Fig. 5 B). These findings were confirmed by in situ evaluation of CAM tissue sections, revealing that the phospho-Raf 338 staining was primarily associated with bFGF-stimulated blood vessels (Fig. 5 C).


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)

Integrins αvβ3 and αvβ5 differentially influence PAK activity during angiogenesis. (A) 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. 1 h before tissue excision, function-blocking antibodies directed against integrin αvβ3 or αvβ5 were i.v. injected. Endogenous PAK was immunoprecipitated from equivalent amounts of total protein and subjected to a kinase assay using myelin basic protein as a substrate, electrophoresed, and transferred to nitrocellulose as described in Materials and methods. The above blot was probed with an anti-PAK antibody as a loading control. (B) Chick CAMs were treated as above with the exception that total lysates were probed with an antibody directed specific to c-Raf phosphorylated at serine 338. The above blot was probed with an anti-c-Raf antibody as a loading control. (C) 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 c-Raf phosphorylated at serine 338. Bar, 50 μm.
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Related In: Results  -  Collection

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fig5: Integrins αvβ3 and αvβ5 differentially influence PAK activity during angiogenesis. (A) 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. 1 h before tissue excision, function-blocking antibodies directed against integrin αvβ3 or αvβ5 were i.v. injected. Endogenous PAK was immunoprecipitated from equivalent amounts of total protein and subjected to a kinase assay using myelin basic protein as a substrate, electrophoresed, and transferred to nitrocellulose as described in Materials and methods. The above blot was probed with an anti-PAK antibody as a loading control. (B) Chick CAMs were treated as above with the exception that total lysates were probed with an antibody directed specific to c-Raf phosphorylated at serine 338. The above blot was probed with an anti-c-Raf antibody as a loading control. (C) 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 c-Raf phosphorylated at serine 338. Bar, 50 μm.
Mentions: The finding that αvβ3 appears to regulate c-Raf, but not Ras activity in response to bFGF prompted us to evaluate how αvβ3 contributes to c-Raf activation. Recently, we found that bFGF-mediated angiogenesis is dependent on PAK-1 signaling (Kiosses et al., 2002). Importantly, PAK-1 phosphorylates c-Raf on serine 338, leading to its activation (Zang et al., 2002). To evaluate whether αvβ3 signaling is linked to PAK-1's role in angiogenesis, CAMs stimulated with bFGF or VEGF and treated with anti-αvβ3 or -αvβ5 were lysed and analyzed for PAK-1 activity. Although both growth factors stimulated PAK activity, only anti-αvβ3 was able to suppress this response (Fig. 5 A). Accordingly, direct evaluation of the tissue lysates revealed that bFGF (but not VEGF) induced an αvβ3-dependent phosphorylation of c-Raf at its PAK phosphorylation site, S338 (Fig. 5 B). These findings were confirmed by in situ evaluation of CAM tissue sections, revealing that the phospho-Raf 338 staining was primarily associated with bFGF-stimulated blood vessels (Fig. 5 C).

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