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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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Src requirement for Raf-ERK activation during VEGF-induced (but not bFGF-induced) angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF or VEGF for 20 h, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were 1.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 or αvβ5 as indicated. Endogenous Src was immunoprecipitated and subjected to an in vitro kinase assay using a FAK-GST fusion protein as a substrate, electrophoresed, and anti-Src antibody was used as a loading control as described in Materials and methods. (B) 10-d-old chick CAMs were treated as described above with the exception that after excision, c-Raf was immunoprecipitated from the tissue extracts and probed with an antibody directed against phosphorylated tyrosine 340 on c-Raf. The above blot was then stripped and probed with an anti-c-Raf antibody as a loading control. (C) Chick CAMs were stimulated as described above with the exception that filter paper disks on the CAM were saturated with either the Src inhibitor PP1 or RCAS-Src251 (inactive Src), followed by blotting for phospho-Raf 340 or anti-c-Raf. (D) Chick CAMs were stimulated as described above with the exception that lysates were probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.
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fig4: Src requirement for Raf-ERK activation during VEGF-induced (but not bFGF-induced) angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF or VEGF for 20 h, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were 1.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 or αvβ5 as indicated. Endogenous Src was immunoprecipitated and subjected to an in vitro kinase assay using a FAK-GST fusion protein as a substrate, electrophoresed, and anti-Src antibody was used as a loading control as described in Materials and methods. (B) 10-d-old chick CAMs were treated as described above with the exception that after excision, c-Raf was immunoprecipitated from the tissue extracts and probed with an antibody directed against phosphorylated tyrosine 340 on c-Raf. The above blot was then stripped and probed with an anti-c-Raf antibody as a loading control. (C) Chick CAMs were stimulated as described above with the exception that filter paper disks on the CAM were saturated with either the Src inhibitor PP1 or RCAS-Src251 (inactive Src), followed by blotting for phospho-Raf 340 or anti-c-Raf. (D) Chick CAMs were stimulated as described above with the exception that lysates were probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.

Mentions: Previous reports have documented that VEGF-mediated (but not bFGF-mediated) angiogenesis depends on the activity of Src family kinases (Eliceiri et al., 1999) and αvβ5 (Friedlander et al., 1995). To evaluate whether αvβ5 signaling is linked to Src's role in angiogenesis, CAMs stimulated with bFGF or VEGF and treated with anti-αvβ3 or -αvβ5, respectively, were lysed and analyzed for Src activity. Although both growth factors stimulated Src activity, neither antibody was able to suppress this response (Fig. 4 A), which is consistent with previous findings that Src activity is upstream of integrin signaling on the VEGF pathway (Eliceiri et al., 2002). Notably, Src can also regulate c-Raf activation by phosphorylating c-Raf on tyrosines 340/341 (Fabian et al., 1993), a site phosphorylated in response to stimulation by VEGF, but not bFGF (Alavi et al., 2003). To evaluate the role of αvβ5 in VEGF-induced c-Raf phosphorylation, CAMs stimulated with bFGF or VEGF were treated with either anti-αvβ3 or -αvβ5, respectively, and c-Raf was analyzed for phosphorylation of tyrosines 340/341 (Fig. 4 B). In agreement with earlier reports (Alavi et al., 2003), VEGF (but not bFGF) induced phosphorylation of Raf on tyrosines 340/341. However, this phosphorylation event was completely blocked by inhibition of integrin αvβ5. To confirm the role of Src in VEGF-induced c-Raf activation, CAMs stimulated with bFGF or VEGF were transduced with RCAS-Src251 or treated with the Src inhibitor PP1 and subsequently analyzed for phosphorylation of c-Raf at tyrosines 340/341 (Fig. 4 C). Consistent with a direct role for Src in VEGF-mediated c-Raf activation, VEGF-induced (but not bFGF-induced) phosphorylation of c-Raf on tyrosines 340/341 was blocked by treatment of tissues with Src251 or PP1. Furthermore, VEGF (but not bFGF) induced ERK activity that was completely abolished by Src251 or PP1 (Fig. 4 D). These findings reveal that VEGF/αvβ5 selectively use Src to phosphorylate c-Raf in vivo, leading to 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)

Src requirement for Raf-ERK activation during VEGF-induced (but not bFGF-induced) angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF or VEGF for 20 h, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were 1.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 or αvβ5 as indicated. Endogenous Src was immunoprecipitated and subjected to an in vitro kinase assay using a FAK-GST fusion protein as a substrate, electrophoresed, and anti-Src antibody was used as a loading control as described in Materials and methods. (B) 10-d-old chick CAMs were treated as described above with the exception that after excision, c-Raf was immunoprecipitated from the tissue extracts and probed with an antibody directed against phosphorylated tyrosine 340 on c-Raf. The above blot was then stripped and probed with an anti-c-Raf antibody as a loading control. (C) Chick CAMs were stimulated as described above with the exception that filter paper disks on the CAM were saturated with either the Src inhibitor PP1 or RCAS-Src251 (inactive Src), followed by blotting for phospho-Raf 340 or anti-c-Raf. (D) Chick CAMs were stimulated as described above with the exception that lysates were probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.
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fig4: Src requirement for Raf-ERK activation during VEGF-induced (but not bFGF-induced) angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF or VEGF for 20 h, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were 1.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 or αvβ5 as indicated. Endogenous Src was immunoprecipitated and subjected to an in vitro kinase assay using a FAK-GST fusion protein as a substrate, electrophoresed, and anti-Src antibody was used as a loading control as described in Materials and methods. (B) 10-d-old chick CAMs were treated as described above with the exception that after excision, c-Raf was immunoprecipitated from the tissue extracts and probed with an antibody directed against phosphorylated tyrosine 340 on c-Raf. The above blot was then stripped and probed with an anti-c-Raf antibody as a loading control. (C) Chick CAMs were stimulated as described above with the exception that filter paper disks on the CAM were saturated with either the Src inhibitor PP1 or RCAS-Src251 (inactive Src), followed by blotting for phospho-Raf 340 or anti-c-Raf. (D) Chick CAMs were stimulated as described above with the exception that lysates were probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.
Mentions: Previous reports have documented that VEGF-mediated (but not bFGF-mediated) angiogenesis depends on the activity of Src family kinases (Eliceiri et al., 1999) and αvβ5 (Friedlander et al., 1995). To evaluate whether αvβ5 signaling is linked to Src's role in angiogenesis, CAMs stimulated with bFGF or VEGF and treated with anti-αvβ3 or -αvβ5, respectively, were lysed and analyzed for Src activity. Although both growth factors stimulated Src activity, neither antibody was able to suppress this response (Fig. 4 A), which is consistent with previous findings that Src activity is upstream of integrin signaling on the VEGF pathway (Eliceiri et al., 2002). Notably, Src can also regulate c-Raf activation by phosphorylating c-Raf on tyrosines 340/341 (Fabian et al., 1993), a site phosphorylated in response to stimulation by VEGF, but not bFGF (Alavi et al., 2003). To evaluate the role of αvβ5 in VEGF-induced c-Raf phosphorylation, CAMs stimulated with bFGF or VEGF were treated with either anti-αvβ3 or -αvβ5, respectively, and c-Raf was analyzed for phosphorylation of tyrosines 340/341 (Fig. 4 B). In agreement with earlier reports (Alavi et al., 2003), VEGF (but not bFGF) induced phosphorylation of Raf on tyrosines 340/341. However, this phosphorylation event was completely blocked by inhibition of integrin αvβ5. To confirm the role of Src in VEGF-induced c-Raf activation, CAMs stimulated with bFGF or VEGF were transduced with RCAS-Src251 or treated with the Src inhibitor PP1 and subsequently analyzed for phosphorylation of c-Raf at tyrosines 340/341 (Fig. 4 C). Consistent with a direct role for Src in VEGF-mediated c-Raf activation, VEGF-induced (but not bFGF-induced) phosphorylation of c-Raf on tyrosines 340/341 was blocked by treatment of tissues with Src251 or PP1. Furthermore, VEGF (but not bFGF) induced ERK activity that was completely abolished by Src251 or PP1 (Fig. 4 D). These findings reveal that VEGF/αvβ5 selectively use Src to phosphorylate c-Raf in vivo, leading to 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