RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G + -bacterial toxins

Abstract

Disruption of the endothelial barrier in response to Gram positive (G+) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G+ toxin-induced microvascular permeability. We found that the G+ toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G+-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G+-toxins.

RAC1 nitration at Y32 IS involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury

“…NMR and X-ray crystal structures for Rho family GTPases propose that the Cys18 thiol in the GXXXXGK(S/T)C motif is accessible for solvent and suggest reactive oxygen species and reactive nitrogen species possibly target the Cys18 thiol [ [35] , [36] , [37] ]. Further, we have shown that S-nitrosylation of RhoA attenuates the activity of RhoA[ 38 ]. The effect of S-nitrosylation on Rac1 is unresolved but we speculate that as nitration activates while S-nitrosylation inhibits RhoA that S-nitrosylation may stimulate Rac1 activity.…”

Hsp70 Suppresses Mitochondrial Reactive Oxygen Species and Preserves Pulmonary Microvascular Barrier Integrity Following Exposure to Bacterial Toxins

“…Pneumolysin has been well documented to disrupt barrier function in HLMVEC and increase endothelial permeability ( 12 , 13 , 41 , 42 ). To determine the effect of Hsp70 on barrier function, HLMVECs were transduced with adenoviruses encoding Hsp70 (pAd Hsp70-GFP, 60MOI) or GFP (Control, 60MOI) overnight and then transferred to an ECIS plate.…”

“…Pneumolysin and other cholesterol-dependent pore-forming cytolysins from G + -bacteria such as listeriolysin O have been shown to form plasma membrane pores that stimulate calcium entry and promote disruption of the endothelial barrier ( 12 , 13 , 41 , 42 , 47 ). Multiple mechanisms have been proposed, including activation of PKC, disruption of NO signaling, arginase induction, and inhibition of ENaC.…”

Activation of the mechanosensitive Ca2+ channel TRPV4 induces endothelial barrier permeability via the disruption of mitochondrial bioenergetics

“…A number of molecular mechanisms have been identified in VILI including Ca 2+ dependent pathways, activation of protein kinases (including PKC and Rho kinase) and the modulation of eNOS activity [ 13 ], and an excessive ROS generation [ [14] , [15] , [16] , [17] ]. Interestingly, our recent study demonstrated that PKC-dependent phosphorylation of eNOS T495 leads to enzyme uncoupling, increased peroxynitrite production, and barrier disruption [ 18 ].…”

“…Further investigations also revealed that PKC dependent phosphorylation of eNOS at Thr495, increased mitochondrial derived ROS [ 19 ]. It is widely accepted that calcium entry into EC is necessary for barrier disruption [ 14 ]. As eNOS uncoupling can be catalyzed by PKCα [ 16 ] this suggest a link between increases in intracellular [Ca 2+ ] and eNOS uncoupling and the EC barrier permeability associated with VILI.…”