Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Non-Muscle Myosin II
Rationale: Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells (ECs), apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 is required for apical junction clearance, as well as for regulation of Rho GTPase activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis.
Objective: The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis.
Methods and Results: Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of EC apical junctions during vascular tubulogenesis depends on Ras interacting protein 1 (Rasip1), as well as the GTPase Cdc42 and the kinase Pak4. Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all block EC tubulogenesis. By contrast, inactivation of RhoA signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII either prior to, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles.
Conclusions: Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.
- Received May 15, 2016.
- Revision received July 29, 2016.
- Accepted August 2, 2016.