PKC-Dependent Phosphorylation of eNOS at T495 Regulates eNOS Coupling and Endothelial Barrier Function in Response to G+ -Toxins

Abstract

Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G+-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.

Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction

“…PLY reduces endothelial barrier function in part by means of activating protein kinase C-α, which in turn impairs NO generation by endothelial nitric oxide synthase (eNOS) (3, 4), which was shown to be required for basal barrier function (40). Increased Ca 2+ influx can also mobilize calmodulin, which activates CaMKII.…”

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore‐forming toxins

“…Pathogenic Escherichia coli can target Gp96 to promote nitric oxide production, Ca 2+ oscillations and activation of cellular kinases such as PKC-a and FAK, which disrupts endothelial cell junctions and mediates bacterial internalization [40]. LLO and other PFTs trigger such events, which were associated with increased tissue damage in vivo [41,42], and NMHCIIA protects the epithelial barrier in vivo [43]. Interestingly, Lm can interact with Gp96 via its surface protein Vip, which promotes bacterial internalization [18].…”

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

“…Dysfunction of the endothelial barrier arises from changes in cell behavior in response to signaling events that promote contraction, dissolution of adhesive complexes between cells and cell death. Many signaling pathways are involved including calcium, MLC (myosin light chain)-dependent mechanisms [4], cytoskeletal rearrangements [5], disassembly of junctional proteins between cells [6], activation of PKC (protein kinase C) [7], alteration of nitric oxide (NO) signaling [8] and numerous others [9,10]. Unresolved disruption of the endothelial barrier results in the inappropriate loss of fluid from the vasculature and extensive pulmonary edema which are the major complications of acute respiratory distress syndrome (ARDS) and CAP [11].…”