Extracellular beta-nicotinamide adenine dinucleotide (beta-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement

Abstract

Extracellular beta-NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5-trisphate and cAMP. Recently, beta-NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since beta-NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing beta-NAD-activated purinergic receptors upon beta-NAD stimulation. Our data demonstrate that extracellular beta-NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration-dependent manner indicating endothelial barrier enhancement. Importantly, beta-NAD significantly attenuated thrombin-induced EC permeability as well as the barrier-compromising effects of Gram-negative and Gram-positive bacterial toxins representing the barrier-protective function of beta-NAD. Immunofluorescence microscopy reveals more pronounced staining of cell-cell junctional protein VE-cadherin at the cellular periphery signifying increased tightness of the cell-cell contacts after beta-NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in beta-NAD-induced TER increase. beta-NAD-treatment attenuates the lipopolysaccharide (LPS)-induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP-dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in beta-NAD-induced EC barrier enhancement. With these results, we conclude that beta-NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1- and MLCP- dependent signaling pathways.

Keywords: EPAC1; Gq protein; Gs protein; HPAEC; P2Y antagonists; P2Y1 and P2Y11 receptors; Rac1; cAMP.

Figure 1

Extracellular β-NAD enhances barrier function of HPAEC monolayers and increases VE-cadherin presentation in cell-cell contacts. (A) Dose-dependent TER response of β-NAD. HPAEC were challenged with increasing concentration of β-NAD (10–100 μM). Data are representative of several independent experiments (minimum of three) (*p < 0.05 compared with control). (B) Immunofluorescence staining of VE-cadherin in quiescent and β-NAD-stimulated HPAEC monolayers. The cells were treated with 50 μM β-NAD for 30 min, then fixed and immunostained using VE-cadherin antibody. Appreciably more VE-cadherin was recruited to the area of cell-cell junctions after β-NAD treatment. Arrows indicate overlapping edges of neighboring cells. (C) Quantification of the surface area of the cell-cell interface. The percentage of total cell-surface area occupied by VE-cadherin-positive cell-cell junctions was calculated for 20 cells in each group. The graph demonstrates that β-NAD induced a significant increase in cell-cell interface surface area as a percentage of total cell surface area (*p < 0.05 compared with control). The box and Whiskers plot show the means (lines at the box centers, 17.42% and 61.03% for control and β-NAD-treated cells respectively).

Figure 2

Expression of β-NAD-activated purine receptors P2Y1 and P2Y11 on mRNA and protein levels in HPAEC. (A) The receptor mRNA expressions were examined by Real-Time RT-PCR. Data were calculated relative to internal housekeeping gene (18S rRNA) and are expressed as fold change compared to control ± SEM (n=4). (B) The cell lysates obtained from HPAEC were analyzed by SDS-PAGE followed by immunoblotting using rabbit polyclonal antibodies against P2Y1 and P2Y11. Position of 40 kDa protein marker is shown by arrow. Immunoblotting of β-actin was used as a loading control.

Figure 3

Inhibitory analysis (selective antagonists and siRNA-mediated depletion) of the involvement of P2Y1 and P2Y11 receptors in β-NAD-stimulated TER increase. (A) HPAEC were pretreated with either 10 μM MRS2279 (P2Y1 antagonist) or 1 μM NF157 (P2Y11 antagonist) for 30 min prior β-NAD stimulation and used in ECIS assay. Data are representative of several independent experiments (minimum of three) (*p < 0.05 compared with control). (B) RT-PCR analysis of the expression of P2Y1 and P2Y11 mRNAs in the cells treated with scrambled and receptor-specific siRNA. siRNA approach was proved to be very effective in the depletion of P2Y1 and P2Y11 expression. Expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as a loading control. (C, D) Depletions of P2Y11 (C) or P2Y1 (D) receptors negatively affect β-NAD-mediated TER response. HPAEC plated in ECIS arrays were transfected with respective scrambled (nsRNA) and siRNA as described in Materials and Methods. 48 hrs after transfection, the cells were used in ECIS assay in the presence or absence of β-NAD. Time-points of β-NAD addition are indicated by arrows. Data are representative of several independent experiments (minimum of three) (*p < 0.05 compared with control).

Figure 4

Extracellular β-NAD protects HPAEC monolayers from barrier-disruptive effects of thrombin and Gram-negative and Gram-positive bacterial toxins, lipopolysaccharide (LPS) and pneumolysin (PLY). HPAEC plated in ECIS arrays were challenged with either 100 nM thrombin (A) or 100 nM LPS (B) or 31.2 ng/ml PLY (C). The challengers were added either alone or in mixture with 50 μM β-NAD. β-NAD consistently prevented HPAEC barrier dysfunction in the cells treated with thrombin or PLY and significantly protected barrier integrity in the cells treated with LPS. Data are representative of several independent experiments (minimum of three) (*p < 0.05 compared with control).

Figure 5

Molecular mechanisms of β-NAD-activated endothelial barrier enhancement in HPAEC. (A) β-NAD treatment activated cAMP-dependent PKA. HPAEC were pretreated with either vehicle or PKA-specific inhibitor, 5 μM H-89, for 30 min and then stimulated with 50 μM β-NAD in TER measurement assay. The inhibitor pretreatment significantly attenuated β-NAD-dependent increase in TER. (B) cAMP-activated EPAC1 is involved in β-NAD-activated TER response. HPAEC plated in ECIS arrays were transfected with either scrambled (nsRNA) or EPAC1-specific siRNA as described in Materials and Methods. The cells were stimulated with 50 μM β-NAD in TER measurement assay. Successful depletion of EPAC1 led to a significant decrease of β-NAD-activated TER response. (C) Downstream target of PKA/EPAC1 pathways, Rac1, is activated in HPAEC upon β-NAD stimulation. The cells treated with 50 μM β-NAD for time periods indicated were used in G-LISA assay as described in Materials and Methods to estimate the levels of activated Rac1. Data obtained demonstrate a rapid and dramatic elevation of Rac1-activity in β-NAD-stimulated cells. Data are representative of several independent experiments (minimum of three) (*p < 0.05 compared with control).

Figure 6

Effect of cytoskeletal alterations on β-NAD-induced endothelial cell barrier protection. (A) HPAEC were pretreated with either vehicle or cytochalasin B for 30 min and then stimulated with 50 μM β-NAD in TER measurement assay. Actin depolymerization decreased TER and completely prevented the effect of β-NAD on TER. (B) HPAEC were pretreated with either vehicle or the microtubule-disrupting agent, nocodazole, for 30 min and then stimulated with 50 μM β-NAD. Disruption of microtubules decreased TER, but failed to alter β-NAD-induced increase in TER. Results are presented as mean ± SE and derived from three independent experiments (*p < 0.05 compared with control). (C) β-NAD treatment decreases myosin light chain (MLC) phosphorylation stimulated by LPS. HPAEC treated by either vehicle or LPS alone, or LPS/β-NAD mixture for 4 hrs were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phosphoMLC antibody. Immunoblotting with anti-MLC antibody was used as a loading control. Data obtained indicate that barrier-protective mechanism of β-NAD may be realized via stimulation of MLC phosphatase activity, decreasing phosphoMLC levels and preventing, therefore, actin stress fiber formation.

Figure 7

Schematic representation of putative EC barrier-protective molecular mechanisms activated by extracellular β-NAD.