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

FGF and VEGF require and stimulate Ras, Raf, and ERK activity during angiogenesis in cooperation with integrins αvβ3 and αvβ3. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-Ras T17N (inactive Ras), RCAS-RafATPμ (inactive c-Raf), or PD98059 (MEK inhibitor) 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 24 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 either bFGF or VEGF for 5 min, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were i.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 (LM609) or αvβ5 (P1F6). Relative Ras, c-Raf, and ERK was determined as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that CAM tissue was excised 20 h after initial exposure to bFGF and VEGF.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172815&req=5

fig1: FGF and VEGF require and stimulate Ras, Raf, and ERK activity during angiogenesis in cooperation with integrins αvβ3 and αvβ3. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-Ras T17N (inactive Ras), RCAS-RafATPμ (inactive c-Raf), or PD98059 (MEK inhibitor) 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 24 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 either bFGF or VEGF for 5 min, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were i.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 (LM609) or αvβ5 (P1F6). Relative Ras, c-Raf, and ERK was determined as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that CAM tissue was excised 20 h after initial exposure to bFGF and VEGF.

Mentions: As expected, disruption of Ras, c-Raf, or MEK activity in these tissues suppressed angiogenesis induced with either growth factor (Fig. 1). In the absence of integrin antagonists, the activity of Ras, c-Raf, and ERK was induced within 5 min and remained active for at least 20 h after either bFGF or VEGF stimulation (Fig. 1, B and C). Intravenous administration of anti-αvβ3, either 1 h before growth factor addition, or 1 h before harvest at the 20-h time point, showed no diminution of bFGF-induced early or late Ras activity in CAM tissues (Fig. 1, B and C). However, anti-αvβ3 did prevent the bFGF-induced sustained c-Raf and ERK activity (Fig. 1, B and C). In contrast, i.v. injection of anti-αvβ5 completely blocked VEGF-mediated late Ras and c-Raf, as well as ERK activity (Fig. 1, B and C). Importantly, anti-αvβ3 did not suppress VEGF-induced, nor did anti-αvβ5 suppress bFGF-induced Ras, c-Raf, or ERK signaling (Fig. 1, B and C). These findings suggest that ligation of specific αv integrins differentially regulates bFGF- and VEGF-mediated activation of the Ras-ERK signaling pathway in angiogenic tissue in vivo. In this case, VEGF-mediated (but not bFGF-mediated) activation of Ras depends on the coordinated ligation of a specific αv integrin. Our findings can likely be attributed to a direct effect on blood vessels because VEGF preferentially activates ECs, and αvβ3 is exclusively expressed by ECs within these tissues.


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)

FGF and VEGF require and stimulate Ras, Raf, and ERK activity during angiogenesis in cooperation with integrins αvβ3 and αvβ3. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-Ras T17N (inactive Ras), RCAS-RafATPμ (inactive c-Raf), or PD98059 (MEK inhibitor) 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 24 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 either bFGF or VEGF for 5 min, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were i.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 (LM609) or αvβ5 (P1F6). Relative Ras, c-Raf, and ERK was determined as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that CAM tissue was excised 20 h after initial exposure to bFGF and VEGF.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: FGF and VEGF require and stimulate Ras, Raf, and ERK activity during angiogenesis in cooperation with integrins αvβ3 and αvβ3. (A) 10-d-old chick CAMs were exposed to filter paper disks saturated with RCAS-Ras T17N (inactive Ras), RCAS-RafATPμ (inactive c-Raf), or PD98059 (MEK inhibitor) 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 24 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 either bFGF or VEGF for 5 min, followed by excision and detergent extraction of the tissues. 1 h before excision, the embryos were i.v. injected with 30 μg function-blocking antibodies selective for either integrin αvβ3 (LM609) or αvβ5 (P1F6). Relative Ras, c-Raf, and ERK was determined as described in Materials and methods. (C) Chick CAMs were treated as above with the exception that CAM tissue was excised 20 h after initial exposure to bFGF and VEGF.
Mentions: As expected, disruption of Ras, c-Raf, or MEK activity in these tissues suppressed angiogenesis induced with either growth factor (Fig. 1). In the absence of integrin antagonists, the activity of Ras, c-Raf, and ERK was induced within 5 min and remained active for at least 20 h after either bFGF or VEGF stimulation (Fig. 1, B and C). Intravenous administration of anti-αvβ3, either 1 h before growth factor addition, or 1 h before harvest at the 20-h time point, showed no diminution of bFGF-induced early or late Ras activity in CAM tissues (Fig. 1, B and C). However, anti-αvβ3 did prevent the bFGF-induced sustained c-Raf and ERK activity (Fig. 1, B and C). In contrast, i.v. injection of anti-αvβ5 completely blocked VEGF-mediated late Ras and c-Raf, as well as ERK activity (Fig. 1, B and C). Importantly, anti-αvβ3 did not suppress VEGF-induced, nor did anti-αvβ5 suppress bFGF-induced Ras, c-Raf, or ERK signaling (Fig. 1, B and C). These findings suggest that ligation of specific αv integrins differentially regulates bFGF- and VEGF-mediated activation of the Ras-ERK signaling pathway in angiogenic tissue in vivo. In this case, VEGF-mediated (but not bFGF-mediated) activation of Ras depends on the coordinated ligation of a specific αv integrin. Our findings can likely be attributed to a direct effect on blood vessels because VEGF preferentially activates ECs, and αvβ3 is exclusively expressed by ECs within these tissues.

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