Limits...
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.

Show MeSH

Related in: MedlinePlus

Ras and Raf are differentially regulated by integrins αvβ3 and αvβ5 during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF, VEGF, RCAS-Ras G12V (active Ras), or RCAS-Raf-caax (active c-Raf). After 20 h, embryos were i.v. injected with function-blocking antibodies directed against integrins αvβ3 or αvβ5. After 72 h, blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 24 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ3 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control. (C) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ5 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172815&req=5

fig3: Ras and Raf are differentially regulated by integrins αvβ3 and αvβ5 during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF, VEGF, RCAS-Ras G12V (active Ras), or RCAS-Raf-caax (active c-Raf). After 20 h, embryos were i.v. injected with function-blocking antibodies directed against integrins αvβ3 or αvβ5. After 72 h, blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 24 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ3 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control. (C) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ5 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.

Mentions: To further establish the role αv integrins play in Ras-Erk signaling and angiogenesis, unstimulated CAMs were transduced with active forms of Ras (G12V-Ras) or c-Raf (Raf-caax), which promoted a strong angiogenic response in these tissues (Fig. 3 A). CAMs stimulated with active Ras or c-Raf were then treated with either anti-αvβ3 or -αvβ5 and analyzed for Ras or c-Raf activity after 20 h, or measured for angiogenesis after 72 h. Expression of active Ras or c-Raf induced an equivalent or greater angiogenic response compared with that seen with bFGF or VEGF (Fig. 3 A). Although anti-αvβ3 blocked bFGF as well as Ras-mediated angiogenesis, it had little effect on that induced with c-Raf, suggesting that αvβ3 potentiates signaling to ERK downstream of Ras, but at or upstream of c-Raf. In contrast, anti-αvβ5 blocked VEGF-induced angiogenesis, yet had no effect on angiogenesis induced with activated Ras or c-Raf, suggesting that the anti-angiogenic effects of anti-αvβ5 was upstream of both Ras and c-Raf. These results are consistent with the finding that anti-αvβ5 blocks VEGF-mediated Ras activity, whereas anti-αvβ3 disrupts bFGF-mediated c-Raf activation, but fails to block Ras activation.


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)

Ras and Raf are differentially regulated by integrins αvβ3 and αvβ5 during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF, VEGF, RCAS-Ras G12V (active Ras), or RCAS-Raf-caax (active c-Raf). After 20 h, embryos were i.v. injected with function-blocking antibodies directed against integrins αvβ3 or αvβ5. After 72 h, blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 24 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ3 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control. (C) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ5 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172815&req=5

fig3: Ras and Raf are differentially regulated by integrins αvβ3 and αvβ5 during bFGF- and VEGF-induced angiogenesis. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with either bFGF, VEGF, RCAS-Ras G12V (active Ras), or RCAS-Raf-caax (active c-Raf). After 20 h, embryos were i.v. injected with function-blocking antibodies directed against integrins αvβ3 or αvβ5. After 72 h, blood vessels were enumerated by counting vessel branch points in a double-blinded manner. Each bar represents the mean ± SEM of 24 replicates. *, P < 0.05 relative to control; **, P < 0.05 relative to treatment. (B) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ3 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control. (C) Chick CAMs were treated as above with the exception that antibodies directed against integrin αvβ5 were injected after 19 h of growth-factor stimulation followed by excision and detergent extraction 1 h later. Lysates were then electrophoresed and probed with antibodies directed against the active, phosphorylated form of ERK or an anti-ERK antibody as a loading control.
Mentions: To further establish the role αv integrins play in Ras-Erk signaling and angiogenesis, unstimulated CAMs were transduced with active forms of Ras (G12V-Ras) or c-Raf (Raf-caax), which promoted a strong angiogenic response in these tissues (Fig. 3 A). CAMs stimulated with active Ras or c-Raf were then treated with either anti-αvβ3 or -αvβ5 and analyzed for Ras or c-Raf activity after 20 h, or measured for angiogenesis after 72 h. Expression of active Ras or c-Raf induced an equivalent or greater angiogenic response compared with that seen with bFGF or VEGF (Fig. 3 A). Although anti-αvβ3 blocked bFGF as well as Ras-mediated angiogenesis, it had little effect on that induced with c-Raf, suggesting that αvβ3 potentiates signaling to ERK downstream of Ras, but at or upstream of c-Raf. In contrast, anti-αvβ5 blocked VEGF-induced angiogenesis, yet had no effect on angiogenesis induced with activated Ras or c-Raf, suggesting that the anti-angiogenic effects of anti-αvβ5 was upstream of both Ras and c-Raf. These results are consistent with the finding that anti-αvβ5 blocks VEGF-mediated Ras activity, whereas anti-αvβ3 disrupts bFGF-mediated c-Raf activation, but fails to block Ras activation.

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