Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: Molecular mechanisms and barrier-protective role

Abstract

We have previously shown that Gs-coupled adenosine receptors (A2a) are primarily involved in adenosine-induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine-induced Rac1 activation and EC barrier enhancement is mediated by Gs-dependent stimulation of cAMP-dependent Epac1-mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively-active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine-induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine-induced increase in TER. We next examined the role of cAMP-activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine-induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine-induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine-induced increases in TER and Tiam1 depletion had no effect on adenosine-induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine-induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP-dependent Epac1/Vav2/Rap1-mediated signaling.

Keywords: Rac1; adenosine; barrier protection; pulmonary endothelium; small GTPase.

Figure 1.. Rac1 signaling is involved in HPAEC barrier regulation.

To monitor the effect of Rac1 mutants on the resistance of HPAECs, cells were cultured on ECIS plate until they reached 80% confluence and incubated with basal medium for 8 hours before infection. Each well was infected with 1000 MOI of either vector (control), catalytically active (C/A) or dominant negative (D/N) Rac1 mutant adenoviral constructs. After overnight incubation with adenovirus, the medium was replaced with fresh complete EBM-2 medium containing 5% FBS, and the change of resistance was recorded for 20 hours. Normalized resistance was calculated and plotted as a function of time. The inserted western blot data shows the increased expression of Rac1 induced by infection with adenoviral constructs, and GAPDH was used as a loading control. *** p< 0.0001 vs. control; n=3.

Figure 2.

A. Adenosine stimulates Rac1 activity in HPAEC. To measure Rac1 activity in adenosine- treated HPAEC, cells were harvested at different time points (5, 10, 15, and 20 min) and Rac1 G-LISA Activation Assay Kit was used according to the manufacturer’s instructions. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured, n=4; mean +/− SEM * p<0.05 compared to 0 min control. B and C. Adenosine decreases MLC, but not MYPT1 phosphorylation. HPAEC were treated with adenosine at different time points. Cells were harvested and western blots were performed to detect changes in phosphorylated MLC (Thr18/Ser19) and MYPT1 (Thr 853) levels (panels B and C, respectively). Actin was used as a loading control. Bar graphs show the changes of phospho- MYPT1 and diphospho-MLC quantified with scanning densitometry. Results are expressed as Mean and SEM (n=4 experiments). *<0.05 vs. control.

Figure 3.. Attenuating Rac1 activity mitigates the increase in TER induced by adenosine in HPAEC.

To monitor the effect of D/N Rac1 on adenosine-induced TER increase of HPAECs, cells were infected with 1000 MOI of either vector (control) or D/N Rac1. After 24 hour incubation in complete cell culture medium, the change of resistance in the response to adenosine (50 μM) was measured. n=4; mean +/− SEM; **p < 0.01 vs. control/Ado. The arrow indicates the time that adenosine was added.

Figure 4.. Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells.

A. HPAEC were treated with Epac1 specific siRNA or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p<0.05 vs. Non-siRNA/Ado. B. Effect of adenosine on Rac1 activation in Epac1 depleted cells. HPAEC were transfected with Epac1 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; * p<0.05 vs. Non-siRNA/0 min, **P < 0.01 vs. Non-siRNA/0 min, # p<0.05 vs. Epac1-siRNA/0 min, ##p < 0.01 vs. Epac1-siRNA/0 min.

Figure 5.. Depletion of Vav2 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells.

A. HPAEC were treated with Vav2, specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in the TER measurement assay. Depletion of Vav2 significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM;*** p< 0.0001 vs. Non-siRNA/Ado B. Effect of adenosine on Rac1 activation in Vav2 depleted cells. HPAEC were transfected with Vav2 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p < 0.01 vs. Non-siRNA/0 min.

Figure 6.. Depletion of Rap1a attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells.

A. HPAEC were treated with Rap1a specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1a significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; **p < 0.01 vs. Non-siRNA/Ado. B. Effect of adenosine on Rac1 activation in Rap1a depleted cells. HPAEC were transfected with Rap1a specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p < 0.01 vs. Non-siRNA/0 min.

Figure 7.. Depletion of Rap1b modestly affects adenosine-induced increase in TER while substantially decreases Rac1 activation in pulmonary endothelial cells.

A. HPAEC were treated with Rap1b specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1b modestly affected maximal adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine in Rac1 activation in Rap1b depleted cells. HPAEC were transfected with Rap1b specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p < 0.01 vs. Non-siRNA/ 0 min; ##p < 0.01 vs. Rap1b-siRNA/0 min.

Figure 8.. Tiam1 depletion has no effect on adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells.

A. HPAEC were treated with Tiam1 specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Tiam1 did not affect adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine on Rac1 activation in Tiam1 depleted cells. HPAEC were transfected with Tiam1 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **P < 0.01 vs. Non-siRNA/ 0 min; #p < 0.05 vs. Tiam1-siRNA/0 min, ##p < 0.01 vs. Tiam1-siRNA/0 min.

Figure 9.. Schematic representation of adenosine-induced Rac1 signaling.

Rac1 activity is directly linked to EC barrier enhancement. Barrier protective effect of adenosine involved in Rac1 activation. Adenosine induces Rac1 activation and EC barrier enhancement, at least in part, via cAMP-dependent Epac1/Vav2/Rap1a-mediated signaling