Suppression of RhoA Activity by Focal Adhesion Kinase-induced Activation of p190RhoGAP

Abstract

The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.

Role of G protein-coupled receptors in inflammation

“…Activation of RhoA is responsible for stress fiber formation, and increased calcium flux triggers other signaling pathways which ultimately lead to myosin light chain-dependent contraction of endothelial cells [63] . This process is reversible, and focal adhesion kinase plays a role in the reversal of the increased vascular permeability [64] . Thrombin-induced increase in vascular endothelial permeability contributes to the edema seen in inflammatory disorders such as acute lung injury [65,66] .…”

The role of cytoskeleton in the regulation of vascular endothelial barrier function

“…One of them links FAK activation to the assembly of cadherin-dependent junctions [Quadri, Battacharya, 2007]. Another implies FAK-induced down-modulation of RhoA activity and stress fiber assembly [Holinstat et al, 2006]. Further studies are required to define how FAK activation can affect barrier function in vivo.…”

Focal adhesion kinase as a regulator of cell tension in the progression of cancer

“…The Rho GAP, GRAF (GTPase regulator associated with FAK), localizes to stress fibers and FAs and binds to the proline-rich domain at the C-terminus of FAK [41,81], thereby directly linking FAK to a mechanism for the downregulation of the Rho pathway. Another Rho GAP, p190RhoGAP, is phosphorylated (and activated) by Src at focal adhesions [82], and, in endothelial cells, FAK associates with and phosphorylates p190RhoGAP upon treatment with thrombin [83,84]. p190RhoGAP failed to localize to focal adhesions in skin keratinocytes in which FAK expression was conditionally ablated, resulting in elevated Rho activity and enhanced stress fibers [64].…”