Tankyrase inhibition interferes with junction remodeling, induces leakiness, and disturbs YAP1/TAZ signaling in the endothelium.

Tankyrase inhibitors are increasingly considered for therapeutic use in malignancies that are characterized by high intrinsic ß-catenin activity. However, how tankyrase inhibition affects the endothelium after systemic application remains poorly understood. In this study, we aimed to investigate how the tankyrase inhibitor XAV939 affects endothelial cell function and the underlying mechanism involved. Endothelial cell function was analyzed using sprouting angiogenesis, endothelial cell migration, junctional dynamics, and permeability using human umbilical vein endothelial cells (HUVEC) and explanted mouse retina. Underlying signaling was studied using western blot, immunofluorescence, and qPCR in HUVEC in addition to luciferase reporter gene assays in human embryonic kidney cells. XAV939 treatment leads to altered junctional dynamics and permeability as well as impaired endothelial migration. Mechanistically, XAV939 increased stability of the angiomotin-like proteins 1 and 2, which impedes the nuclear translocation of YAP1/TAZ and consequently suppresses TEAD-mediated transcription. Intriguingly, XAV939 disrupts adherens junctions by inducing RhoA-Rho dependent kinase (ROCK)-mediated F-actin bundling, whereas disruption of F-actin bundling through the ROCK inhibitor H1152 restores endothelial cell function. Unexpectedly, this was accompanied by an increase in nuclear TAZ and TEAD-mediated transcription, suggesting differential regulation of YAP1 and TAZ by the actin cytoskeleton in endothelial cells. In conclusion, our findings elucidate the complex relationship between the actin cytoskeleton, YAP1/TAZ signaling, and endothelial cell function and how tankyrase inhibition disturbs this well-balanced signaling.

RhoGEF17-An Essential Regulator of Endothelial Cell Death and Growth.

The Rho guanine nucleotide exchange factor RhoGEF17 was described to reside in adherens junctions (AJ) in endothelial cells (EC) and to play a critical role in the regulation of cell adhesion and barrier function. The purpose of this study was to analyze signal cascades and processes occurring subsequent to AJ disruption induced by RhoGEF17 knockdown. Primary human and immortalized rat EC were used to demonstrate that an adenoviral-mediated knockdown of RhoGEF17 resulted in cell rounding and an impairment in spheroid formation due to an enhanced proteasomal degradation of AJ components. In contrast, ß-catenin degradation was impaired, which resulted in an induction of the ß-catenin-target genes cyclin D1 and survivin. RhoGEF17 depletion additionally inhibited cell adhesion and sheet migration. The RhoGEF17 knockdown prevented the cells with impeded cell-cell and cell-matrix contacts from apoptosis, which was in line with a reduction in pro-caspase 3 expression and an increase in Akt phosphorylation. Nevertheless, the cells were not able to proliferate as a cell cycle block occurred. In summary, we demonstrate that a loss of RhoGEF17 disturbs cell-cell and cell-substrate interaction in EC. Moreover, it prevents the EC from cell death and blocks cell proliferation. Non-canonical ß-catenin signaling and Akt activation could be identified as a potential mechanism.

Structure of Galphaq-p63RhoGEF-RhoA complex reveals a pathway for the activation of RhoA by GPCRs.

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.

Spontaneous release of GDP from Gi proteins and inhibition of adenylyl cyclase in cardiac sarcolemmal membranes.

Several studies have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphate transfer-resistant analog, guanosine 5'- O-(2-thiodiphosphate; GDPbetaS). Here, we studied the mechanism underlying this unconventional activation. Adenylyl cyclase activity in sarcolemmal membranes from failing human ventricular myocardium, at a low Mg2+ concentration, decreased rapidly during incubation at 37 degrees C. This decrease in enzyme activity was paralleled by a rapid release of GDP from Gi proteins, amounting to 75% release of total Gi-bound GDP within 10 min at 37 degrees C. In contrast, no GDP release was observed at 4 degrees C, and adenylyl cyclase activity remained stable for up to 20 min at 4 degrees C. GDPbetaS did not alter the initial rates of cyclic AMP formation by the adenylyl cyclase, at either 37 degrees C or 4 degrees C, but almost fully prevented the decrease in enzyme activity occurring at 37 degrees C. Hence, after 10 min of incubation at 37 degrees C, adenylyl cyclase activity in the presence of GDPbetaS was increased by 150%, while no difference in activity was observed at 4 degrees C. Under conditions where adenylyl cyclase is uncoupled from regulation by the inhibitory Gi proteins, i.e., at high concentrations of Mg2+ or Mn2+, adenylyl cyclase activity remained stable even at 37 degrees C and GDPbetaS did not stimulate activity. In conclusion, Gi proteins in sarcolemmal membranes from failing human hearts rapidly release bound GDP. The data, furthermore, suggest that this process results in adenylyl cyclase inhibition by the empty but apparently active Gi proteins.