Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy
Weiquan Zhu, Dallas S. Shi, Jacob M. Winter, Bianca E. Rich, Zongzhong Tong, Lise K. Sorensen, Helong Zhao, Yi Huang, Zhengfu Tai, Tara M. Mleynek, Jae Hyuk Yoo, Christine Dunn, Jing Ling, Jake A. Bergquist, Jackson R. Richards, Amanda Jiang, Lisa A. Lesniewski, M. Elizabeth Hartnett, Diane M. Ward, Alan L. Mueller, Kirill Ostanin, Kirk R. Thomas, Shannon J. Odelberg, Dean Y. Li
Weiquan Zhu, Dallas S. Shi, Jacob M. Winter, Bianca E. Rich, Zongzhong Tong, Lise K. Sorensen, Helong Zhao, Yi Huang, Zhengfu Tai, Tara M. Mleynek, Jae Hyuk Yoo, Christine Dunn, Jing Ling, Jake A. Bergquist, Jackson R. Richards, Amanda Jiang, Lisa A. Lesniewski, M. Elizabeth Hartnett, Diane M. Ward, Alan L. Mueller, Kirill Ostanin, Kirk R. Thomas, Shannon J. Odelberg, Dean Y. Li
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Research Article Cell biology Ophthalmology

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

Abstract

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs — ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

Authors

Weiquan Zhu, Dallas S. Shi, Jacob M. Winter, Bianca E. Rich, Zongzhong Tong, Lise K. Sorensen, Helong Zhao, Yi Huang, Zhengfu Tai, Tara M. Mleynek, Jae Hyuk Yoo, Christine Dunn, Jing Ling, Jake A. Bergquist, Jackson R. Richards, Amanda Jiang, Lisa A. Lesniewski, M. Elizabeth Hartnett, Diane M. Ward, Alan L. Mueller, Kirill Ostanin, Kirk R. Thomas, Shannon J. Odelberg, Dean Y. Li

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Figure 4

The ARF-GEF ARNO is required for VEGFR2 internalization.

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The ARF-GEF ARNO is required for VEGFR2 internalization. (A) VEGF-treate...
(A) VEGF-treated HRMECs were assayed for ARF6 activation using an ARF6-GTP–pulldown assay. (B) ARNO siRNA–treated HRMECs were assayed for VEGF-induced ARF6 activation. (C) ARNO siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2 phosphorylation. (D) ARNO siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for PLCγ, p38, ERK1/2, and MARCKS phosphorylation. (E) ARNO siRNA– or control siRNA–treated HRMECs were labeled with biotin, stimulated with VEGF for 5 minutes, and assayed for internalized VEGFR2. (F) ARNO siRNA– or control siRNA–treated HRMECs were assayed for VEGF-induced cell migration. (G) HRMECs infected with Ad-null, Ad-ARNOWT, or Ad-ARNOE156K were stimulated with VEGF and assayed for VEGFR2 phosphorylation. In A (n = 5) and E (n = 5), geometric means and 95%CIs (error bars) of the ratios (each data point was normalized to its respective untreated control) were calculated, and the ratios were plotted on a logarithmic scale. Statistical significance was assessed using the ratio paired, 2-tailed t test on non-normalized data, and the P value is shown in each graph. In B (n = 3), F (n = 4), and G (n = 3), a 1-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001). All error bars represent the SEM.