RhoJ Regulates α5β1 Integrin Trafficking to Control Fibronectin Remodeling during Angiogenesis

Abstract

Rho GTPases are master regulators of cell shape and cell movement [1]. The archetypal family members RhoA, Rac1 and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and more complex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins -RhoQ and RhoJ [2]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [3,4]. RhoJ is required for angiogenesis [5,6] and has multiple roles in this process [7,8]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [9]. Here we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5β1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5β1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.

Angiogenesis: The Importance of RHOJ-Mediated Trafficking of Active Integrins

“…In agreement with their conclusions on active α5β1 integrins, Sundararaman et al [8] show that RHOJ silencing enhances the ability of ECs to polymerize both endogenous and exogenous soluble FN into an insoluble matrix, while active RHOJ overexpression results in the opposite effect. Since endogenous FN secretion is not affected by either the lack or the constitutive activation of RHOJ, it is conceivable that the effect of RHOJ on FN polymerization in ECs is not direct, rather deriving from the regulation of active α5β1 integrin levels.…”

“…This fine control depends not only on the regulation of integrin allosteric conformation [6], but also endosomal traffic to and from the plasma membrane of both inactive and active integrin conformers via a complex, and only partially characterized, network of functionally distinct pools of cytosolic vesicles [4,7]. In this issue of Current Biology Sundararaman et al [8] unveil how the endothelial small GTPase RHOJ, which is known to control vascular morphogenesis [9,10], acts by selectively regulating the traffic of active, but not inactive α5β1 integrin, thus finely tuning the assembly of FN into polymeric fibrils both in cultured ECs and in vivo, thus allowing physiological angiogenesis to occur (Summarized in Figure 1).…”

“…Sundararaman et al [8] provide novel evidence about the role that the CDC42-related RHO family member RHOJ plays in controlling active α5β1 integrins internalization and post-endocytic traffic in ECs, where this small GTPase localizes in early endosomes (EEs), late endosomes/lysosomes (LEs/LYs), but not PGCs. α5β1 integrin is the main cargo of RHOJ containing vesicles.…”

“…Of note, the Authors also report that, when compared to RHOJ, silencing its close relative CDC42 or overexpressing a constitutively active isoform of it result in fully complementary effects on FN polymerization, suggesting that CDC42 and RHOJ are two closely related small GTPases that may reciprocally inhibit each other to control active α5β1 integrin levels and FN fibrillogenesis. Sundararaman et al [8] identify p21 (RAC1) activated kinase 3 (PAK3) as a shared effector protein for whose binding CDC42 may compete with RHOJ. PAK3 interacts with higher affinity with CDC42 than with RHOJ.…”

RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells

“…This process may also occur in ECs, in which internalized PlexinD1 is ultimately recycled to the plasma membrane after Sema3E stimulation. Given that RhoJ regulates endocytic trafficking of cargo proteins such as transferrin receptor, podocalyxin, and a5b1 integrin (de Toledo et al, 2003;Richards et al, 2015;Sundararaman et al, 2020), it will be interesting to investigate how the binding and release of RhoJ influence the trafficking route of PlexinD1. Of note, Rnd2 also binds to the RBD of PlexinD1 and regulates its R-Ras GAP activity (Uesugi et al, 2009).…”