RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development

Abstract

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. In this study, we demonstrate a novel and critical role for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models led to apoptosis and failures in tube formation. We identified a novel link between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy revealed that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. We show that this colocalization is functionally relevant, because when RhoB was depleted, Akt was excluded from the nucleus and total cellular Akt protein was decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to only be rate limiting for endothelial cell sprouting, we propose that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable.

RhoB facilitates c-Myc turnover by supporting efficient nuclear accumulation of GSK-3

“…SGK localized to the nucleus of ER þ /-MEFs ( Figure 5b). Staining appeared to be concentrated at the outer margins of the nucleus, reminiscent of a pattern that has been reported for nuclear Akt (Adini et al, 2003). Notably, SGK localized to the cytosol as well as the nucleus in ER -/-MEFs (Figure 5b).…”

“…Loss of rhoB enhances c-Myc protein stability and cell proliferation RhoB can modulate trafficking of Akt (Adini et al, 2003), which through its regulation of GSK-3 has an important role in controlling c-Myc stability (Sears et al, 2000). Previous studies have shown RhoB to limit the proliferation of transformed cells Liu et al, 2001a, b).…”

“…Various studies have suggested a link between RhoB and Akt based on the ability of RhoB to (i) form a ternary complex with PRK and PDK1 in HeLa cells (Flynn et al, 2000); (ii) suppress the kinase activity of Akt in human cancer cells (Chen et al, 2000;, and (iii) control the nuclear trafficking of Akt in murine endothelial cells (Adini et al, 2003). Western analysis was performed to examine the status of various elements of the Akt pathway in ER MEFs.…”

“…Mellor and colleagues demonstrated initially that RhoB dictates trafficking of the epidermal growth factor receptor (EGFR) (Mellor et al, 1998;Gampel et al, 1999), acting between late endosomes and multivesicle bodies on the pathway to the lysosome (Wherlock et al, 2004). More recently, RhoB has been shown to regulate trafficking of the Akt and Src kinases (Adini et al, 2003;Sandilands et al, 2004). In the case of Akt, support for some regulatory circuit is suggested by the report that RhoB forms a ternary complex with the Rho effector kinase PRK and the pivotal Akt regulatory kinase PDK1 (Flynn et al, 2000), and by the evidence that Akt downregulates RhoB expression in cancer cells (Jiang et al, 2000), perhaps reflecting a feedback loop.…”

5q– myelodysplastic syndromes: chromosome 5q genes direct a tumor-suppression network sensing actin dynamics

“…RhoB has a critical role in apoptotic responses to DNA damage, and the GTPase was shown to be a target of farnesyltransferase inhibitors (FTIs) (Lebowitz et al, 1997;Prendergast, 2001a, b;Adini et al, 2003). RhoB, like other Ras family members, is post-translationally modified on a C-terminal motif (CAAX) by farnesylation.…”