Lack of adducin impairs the stability of endothelial adherens and tight junctions and may be required for cAMP-Rac1-mediated endothelial barrier stabilization

Abstract

Adducin (Add) is an actin binding protein participating in the stabilization of actin/spectrin networks, epithelial junctional turnover and cardiovascular disorders such as hypertension. Recently, we demonstrated that Add is required for adherens junctions (AJ) integrity. Here we hypothesized that Add regulates tight junctions (TJ) as well and may play a role in cAMP-mediated barrier enhancement. We evaluated the role of Add in MyEnd cells isolated from WT and Add-Knock-Out (KO) mice. Our results indicate that the lack of Add drastically alters the junctional localization and protein levels of major AJ and TJ components, including VE-Cadherin and claudin-5. We also showed that cAMP signaling induced by treatment with forskolin and rolipram (F/R) enhances the barrier integrity of WT but not Add-KO cells. The latter showed no junctional reorganization upon cAMP increase. The absence of Add also led to higher protein levels of the small GTPases Rac1 and RhoA. In vehicle-treated cells the activation level of Rac1 did not differ significantly when WT and Add-KO cells were compared. However, the lack of Add led to increased activity of RhoA. Moreover, F/R treatment triggered Rac1 activation only in WT cells. The function of Rac1 and RhoA per se was unaffected by the total ablation of Add, since direct activation with CN04 was still possible in both cell lines and led to improved endothelial barrier function. In the current study, we demonstrate that Add is required for the maintenance of endothelial barrier by regulating both AJ and TJ. Our data show that Add may act upstream of Rac1 as it is necessary for its activation via cAMP.

Cortactin is in a complex with VE-cadherin and is required for endothelial adherens junction stability through Rap1/Rac1 activation

“…In summary, our data set a precedent to place Cttn as a molecular hub able to interact with different junctional proteins like VE-cadherin and β-catenin to preserve basal endothelial junction stability and on the other hand, support cAMP-mediated barrier fortification by enabling the activation of Rap1 and Rac1. Finally, it is important to note that the functions of Cttn in modulation of cAMP-mediated regulation of small GTPases is in line with previous findings on vasodilator-stimulated phosphoprotein (VASP) 93,94 and adducin 13 . From this, we conclude that a central function of actin binding proteins associated with endothelial AJ is to fine-tune small GTPases such as Rap1, Rac1 and RhoA in response to changes in cAMP levels during inflammation and potentially also to extracellular mechanical cues.…”

“…Next, the tip of the tail was dissected and processed for DNA extraction and genotyping. Subsequently, the heart was extracted and MyEnds were isolated as described previously 13,43 . Briefly, the isolation procedure was performed at room temperature under the laminar flow hood.…”

“…Junctional fragmentation was quantified as previously described 13 . Briefly, after preparation of the images, the threshold value should be set to eliminate the unspecific staining, background.…”

“…Endothelial barrier recovery was investigated using confluent cell monolayers as described before 13 . In short, cells seeded on 8WE10 gold arrays or on gelatin-coated glass coverslips were treated with 2.5 mM EGTA for 30 min.…”

cAMP: A master regulator of cadherin‐mediated binding in endothelium, epithelium and myocardium

“…139 Recently, AKAP2 was identified for cAMP-independent PKA activation, leading to myosin light chain phosphatase (MLCP) stimulation and consequent MLC dephosphorylation resulting in reduced endothelial cell contraction and endothelial barrier preservation. 140 Additionally, actin-binding proteins including vasodilator-stimulated phosphoprotein (VASP), 103,104,106 adducin, 141,142 and cortactin 143 are involved in the modulation of cAMP-mediated Rac1 activity and barrier function. In this context, the role of cortactin was also demonstrated in vivo.…”