PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

Abstract

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 μM forskolin plus 1 μM IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of32P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of ∼23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…However, thrombin did not activate Cdc42 in HUVEC, in contrast to one previous report that described a delayed activation of Cdc42 in immortalized human dermal microvascular endothelial cells 71. It is worth noting that the same immortalized cell line also does not display Rac1 inactivation in response to thrombin 82. When we modulated Rnd3 expression, thrombin‐induced inactivation of Rac1 was affected.…”

Prostaglandins PGE2 and PGI2 promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

“…In addition PG-induced activation of PKA may cause actin cytoskeletal remodeling by Rac-independent mechanisms, for example, via phosphorylation of myosin light chain kinase and reduction of its enzymatic activity [38,65], or via phosphorylation of Rho regulatory protein RhoGDI leading to Rho inhibition [21,38], or via PKA-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) leading to the structural relaxation of the actin cytoskeleton linked to tight junctions via VASP -ZO-1 complex, which result in enhanced endothelial barrier function [52]. Importantly, our studies revealed strong protective effects of PG in the pulmonary EC model of thrombininduced barrier hyperpermeability and in the murine model of ventilator-induced lung injury ( Figure 8).…”

“…Prostaglandin binding to Gs-coupled EP2, EP4 and IP, which represent "relaxant" type of receptors leads to G sdependent activation of adenylate cyclase and elevation of intracellular cAMP levels [18]. Increases in intracellular cAMP levels have been associated with increased endothelial barrier integrity and linked to activation of PKA, which reduces endothelial MLCK activity, decreases pool of phosphorylated MLC, and leads to relaxation of actomyosin complex, stabilization of F-actin filaments and strengthening of cell-matrix adhesions [19][20][21][22]. In contrast, inhibition of basal cAMP/PKA activity increases pulmonary EC leak in part via activation of MAP kinase Erk1,2 [19].…”

“…One potential mechanism of PKA-dependent barrier protection is PKA-mediated phosphorylation of Rho-GDP dissociation inhibitor, a negative regulator of small GTPase Rho, which results in Rho inactivation and blocks Rho-dependent mechanism of EC hyperpermeability [21]. Activation of cAMP/PKA-mediated signaling also has an inhibitory effect on RhoA activity [23] by direct phosphorylation of RhoA [23,24].…”

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

“…However, no prior evidence of the direct phosphorPhosphorylation of phospholipase D1 and the m odulation of its interaction with RhoA by cAMP-dependent protein kinase ylation of PLD by PKA in vivo and in vitro has been presented. Moreover, direct involvement of the phosphorylations of RhoA and/or PLD in the inhibition of their association is not clearly understood although RhoA phosphorylation by PKA was shown to enhance or inhibit the association between RhoA and its binding proteins, such as Rho GDP dissociation inhibitor (RhoGDI) and Rho kinase (Lang et al, 1996;Dong et al, 1998;Forget et al, 2002;Ellerbroek et al, 2003;Qiao et al, 2003).…”