Abstract
Endothelial barrier function is carefully controlled to protect tissues from edema and damage inflicted by extravasated leukocytes. RhoGTPases, in conjunction with myriad regulatory proteins, exert both positive and negative effects on the endothelial barrier integrity. Precise knowledge about the relevant mechanisms is currently fragmented and we therefore performed a comprehensive analysis of endothelial barrier regulation by RhoGTPases and their regulators. Combining RNAi with electrical impedance measurements we quantified the relevance of 270 Rho-associated genes for endothelial barrier function. Statistical analysis identified 10 targets of which six promoted- and four reduced endothelial barrier function upon downregulation. We analyzed in more detail two of these which were not previously identified as regulators of endothelial integrity. We found that the Rac1-GEF (Guanine nucleotide Exchange Factor) TIAM2 is a positive regulator and the Cdc42(Rac1)-GAP (GTPase-Activating Protein) SYDE1 is a negative regulator of the endothelial barrier function. Finally, we found that the GAP SYDE1 is part of a Cdc42-centered signaling unit, also comprising the Cdc42-GEF FARP1 and the Cdc42 effector PAK7 which controls the integrity of the endothelial barrier. In conclusion, using a siRNA-based screen, we identified new regulators of barrier function and found that Cdc42 is a dominant positive regulator of endothelial integrity.
Highlights
The integrity of blood vessels is critical to normal physiology and organ function as it is pivotal for continued circulation and proper distribution and delivery of nutrients and oxygen to the tissues
The ‘on’ state allows interaction with effector proteins and the initiation of downstream signaling. The transition between these states has been intensely studied over the past 25–30 years which uncovered a surprising number of RhoGTPase-regulating molecules that serve to bring Rho proteins in either the ‘on’ or ‘off ’ state[13, 14]. They comprise Guanine nucleotide Exchange Factors (RhoGEFs) which promote the exchange of GDP for GTP, switching the RhoGTPase to ‘on’; and GTPase Activating Proteins (RhoGAPs), which stimulate the intrinsic hydrolysis of GTP, which results in a GDP-bound (‘off ’) RhoGTPase
To understand the mechanism behind the effect of SYDE1 on barrier function, we evaluated which of the major RhoGTPases RhoA, Rac[1] or Cdc[42] were affected after silencing SYDE1
Summary
The integrity of blood vessels is critical to normal physiology and organ function as it is pivotal for continued circulation and proper distribution and delivery of nutrients and oxygen to the tissues. Preservation of endothelial integrity is controlled at multiple levels, in particular at endothelial junctions and the F-actin cytoskeleton[4] This occurs by vessel stabilizing agonists and by intracellular signaling pathways that either enforce barrier properties or balance intracellular events triggered by vessel-disrupting stimuli. The ‘on’ state allows interaction with effector proteins and the initiation of downstream signaling The transition between these states has been intensely studied over the past 25–30 years which uncovered a surprising number of RhoGTPase-regulating molecules that serve to bring Rho proteins in either the ‘on’ or ‘off ’ state[13, 14]. Our combined analysis revealed that a Cdc42-centered signaling unit, comprising the Cdc42GEF FARP1, the Cdc42GAP SYDE1 and Cdc[42] effector proteins, including PAK7, is the most dominant, positive regulator of endothelial integrity
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