Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy
Weiquan Zhu, … , Shannon J. Odelberg, Dean Y. Li
Weiquan Zhu, … , Shannon J. Odelberg, Dean Y. Li
Published October 23, 2017
Citation Information: J Clin Invest. 2017;127(12):4569-4582. https://doi.org/10.1172/JCI91770.
View: Text | PDF
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

×

Figure 5

The ARF-GEF GEP100 is important for VEGFR2 signaling and recycling.

Options: View larger image (or click on image) Download as PowerPoint
The ARF-GEF GEP100 is important for VEGFR2 signaling and recycling. (A) ...
(A) GEP100 siRNA– or control siRNA–treated HRMECs were assayed for VEGF-induced ARF6 activation. (B) GEP100 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2, ERK1/2, and MARCKS phosphorylation. A representative blot from at least 4 independent experiments is shown. (C) GEP100 siRNA– or control siRNA–treated HRMECs were assayed for VEGF-induced cell migration. (D) GEP100 siRNA– or control siRNA–treated HRMECs were labeled with biotin, stimulated with VEGF for 5 minutes, and assayed for internalized VEGFR2. (E) ARF6 siRNA–, ARNO siRNA–, GEP100 siRNA–, or control siRNA–treated HRMECs were stimulated with VEGF for 60 minutes and assayed for VEGFR2 levels. (F) ARF6 siRNA–, ARNO siRNA–, GEP100 siRNA–, or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for colocalization of VEGFR2 with the lysosomal marker LAMP1. Scale bars: 30 μm; original magnification, ×1,200 and additional ×7 (inset). The images in F show a single optical confocal section through an internal region of the HRMECs, and the Z-stacked images are shown in Supplemental Figure 4K. In D, the 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, and the P value is shown in each graph. In A (n = 3), C (n = 4), and E (n = 3), a 1-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance for each time point (**P < 0.01). All error bars represent the SEM.