Wild-type p53 enhances endothelial barrier function by
mediating RAC1 signalling and RhoA inhibition
* , Christiana Dimitropoulou
, Betsy Gregory
, John D. Catravas
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA
School of Medical Diagnostic & Translational Sciences, College of Health Sciences, Old Dominion University, Norfolk, VA, USA
Received: August 8, 2017; Accepted: October 2, 2017
Inﬂammation is the major cause of endothelial barrier hyper-permeability, associated with acute lung injury and acute respiratory distress syn-
drome. This study reports that p53 “orchestrates” the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1
and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1- and P21-acti-
vated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and coﬁlin, in con-
trast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of
phospho-coﬁlin. 17AAG treatment resulted in reduced levels of active coﬁlin. Silencing of coﬁlin pyridoxal phosphate phosphatase (PDXP)
blocked the LPS-induced hyper-permeability, and P53 inhibition reversed the 17AAG-induced PDXP down-regulation. P190RHOGAP suppres-
sion enhanced the LPS-triggered barrier dysfunction in endothelial monolayers. 17AAG treatment resulted in P190RHOGAP induction and
blocked the LPS-induced pMLC2 up-regulation in wild-type mice. Pulmonary endothelial cells from “super p53” mice, which carry additional
p53-tg alleles, exhibited a lower response to LPS than the controls. Collectively, our ﬁndings help elucidate the mechanisms by which p53 oper-
ates to enhance barrier function.
P53 is involved in the regulation of various intracellular cascades
which orchestrate molecular responses to numerous environmental
stimuli. It governs cellular fate, by promoting cell cycle arrest, apop-
tosis or senescence. This transcription factor was discovered
30 years ago as the cellular partner of simian virus 40 large T antigen.
A decade later it became clear that it is a potent tumour suppressor,
which is frequently mutated in humans tumours .
Apart from its role in cancer, P53 is strongly involved in the
defence of vascular endothelium against inﬂammatory insults.
Inﬂammation is a major cause of endothelial barrier dysfunction
and hyper-permeability, leading to acute lung injury (ALI) and acute
respiratory distress syndrome (ARDS) . The development of new
therapeutic strategies against these devastating pathologies has
been slow, and mortality of patients suffering from ARDS remains
around 40% .
We have recently demonstrated that the induction of p53, by
either HSP90 inhibition (by 17AAG) or Nutlin, inhibits the inﬂamma-
tory RhoA pathway  which leads to MLC2 phosphorylation and
subsequent actin stress ﬁbre formation . 17AAG induced p53 by
suppressing the expression of MDM2 and MDM4, the two major p53
negative regulators. Moreover, HSP90 inhibition suppressed the LPS-
induced p53 and MDM2 phosphorylation, modiﬁcations that increase
the rate of p53 proteasomal degradation . In vitro studies on the
effect of p53 silencing on endothelial monolayer permeability have
conﬁrmed that p53 is an essential element for the maintenance of
vascular barrier function .
This study aimed to further investigate the mechanisms which
orchestrate the protective effects of p53 against vascular dysfunction,
focusing on the role of the two major small GTPases which exert
prominent antagonistic roles on endothelial barrier function, namely
Rac1 and RhoA .
Pharmacologic or genetic activation of Rac1 results in vascular
barrier enhancement. Rac1 induces p21-activated kinase (PAK1)
phosphorylation that leads to PAK1 autophosphorylation and activa-
tion. Activated PAK1 phosphorylates LIMK1/2, which, in turn, phos-
phorylates the actin-severing protein coﬁlin at Ser3 and inactivates it
Present address: Department of Basic Pharmaceutical Sciences, School of
Pharmacy, University of Louisiana at Monroe, Monroe, LA 71201
*Correspondence to: Nektarios BARABUTIS, M.Sc., Ph.D.
ª 2018 The Authors.
Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
J. Cell. Mol. Med. Vol 22, No 3, 2018 pp. 1792-1804